Crystalline antibody formulations

ABSTRACT

Described herein are anti-PCSK9 antibody crystals, methods of making such antibody crystals and formulations comprising the antibody crystals.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/326,316, filed Jan. 13, 2017, which is a national stage applicationunder 35 U.S.C. § 371 of International Application No.PCT/US2015/040211, having an international filing date of Jul. 13, 2015,which claims the benefit of, and priority to U.S. ProvisionalApplication No. 62/024,399 filed Jul. 14, 2014, which is incorporated inits entirety by reference herein.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

This application contains, as a separate part of disclosure, a SequenceListing in computer-readable form (filename:A-1860-US-CNT_SequenceListing.txt, created Feb. 24, 2020 which is 42.912bytes in size), which is incorporated by reference in its entirety.

BACKGROUND

Monoclonal antibodies are extensively used as biotherapeutics with anincreasing demand to meet high concentrations of over a 100 mg/ml fordelivery. This presents a challenge for solubility limited proteins viaa subcutaneous route, since the preferred subcutaneous administrationlimit is 1.2 ml (Yang, M. X., Shenoy, B., Disttler, M., Patel, R.,McGrath, M., Pechenov, S., Margolin, A. L. (2003) Crystalline monoclonalantibodies for subcutaneous delivery, PNAS 100, 6934-6939). Developmentof high concentration formulation poses a lot of challenges from aformulation, analytical, stability, manufacturing and drug deliverypoint of view (Shire, S. J., Zahra, S., Liu, J. (2004) Challenges in thedevelopment of high concentration formulations, J. Pharm. Sci. 93,1390-1402). So far, high concentration formulation demands have been metby addition of excipients like amino acids, sugars and salts thatincrease stability, reduce aggregation and viscosity (Shire, supra andJenkins, T. W. (1998) Three solutions of the protein solubility problem,Protein Science 7: 376-382).

Protein crystals are often viewed as only the intermediates to a proteinstructure but they also have an important role from a formulationperspective. Protein molecules in the crystalline form have the lowestentropy thus making them 3-6 kcal/ml more stable than in the liquidstate (Dreuth, J., Haas, C. (1992) Protein crystals and their stability,J. Crystal Growth 122, 107-109). The main advantages of crystallineformulation include high protein concentration, lower viscosity,stability, elimination of frequent dosage due to high concentration andcontrolled release properties (Yang, supra, and Basu, S. K., Govardhan,C. P., Jung, C. W., Margolin, A. L. (2004) Protein crystals for thedelivery of biopharmaceuticals, Expert Opin. Biol. Thera. 4, 301-317).

Crystallization conditions can be manipulated to achieve differentmorphologies for desired controlled release properties (Pechenov, S.,Shenoy, B., Yang, M. X., Basu, S., Margolin, A. L. (2004) Injectablecontrolled release formulations incorporating protein crystals, Journalof Controlled Release 96, 149-158). Insulin crystalline formulationswere first reported in 1920's and today, it is not only the firstrecombinant protein therapeutic approved by the FDA, it is also thefirst approved crystalline protein therapeutic (Hagedorn H. C.; Jensen,B. N.; Krarup, N. B.; Wodstrup, I. Protamine insulinate, (1936) J. Am.Med. Assn. 106, 177-180; Johnson, I. S. (2003) The trials andtribulations of producing the first genetically engineered drug. Nat.Rev. Drug. Discovery 2, 747-751; and Basu, S. K., Govardhan, C. P.,Jung, C. W., Margolin, A. L. (2004) Protein crystals for the delivery ofbiopharmaceuticals, Expert Opin. Biol. Thera. 4, 301-317).Macromolecules are challenging to crystallize due to their inherentflexibility, but, once crystallized, often pose challenges from aformulation and regulatory perspective (Basu, supra, and Jen, A.,Merkle, H. P. (2001) Diamonds in the rough: Protein crystals from aformulation perspective, Pharm. Res. 18, 1483-1488).

SUMMARY OF THE INVENTION

The invention relates to crystals of anti-PCSK9 immunoglobulin type G(IgG) antibodies (more specifically, antibody 21B12) that are suitablefor use in crystalline formulations for parenteral administration;solutions, salts and methods for producing such crystals; methods ofusing such crystals to prepare crystalline formulations for use asmedicaments, and methods of using such crystalline formulations fortreating mammals, specifically humans.

In the crystals or formulations described herein, the anti-PCSK9 IgG cancomprise the heavy and light chain complementary determining regions(CDRs) of antibody, 21B12. Thus, in some embodiments, the antibody is anIgG comprising a light chain complementarity region (CDR) of the CDRL1sequence in SEQ ID NO:9, a CDRL2 of the CDRL2 sequence in SEQ ID NO:9,and a CDRL3 of the CDRL3 sequence in SEQ ID NO:9, and a heavy chaincomplementarity determining region (CDR) of the CDRH1 sequence in SEQ IDNO:5, a CDRH2 of the CDRH2 sequence in SEQ ID NO:5, and a CDRH3 of theCDRH3 sequence in SEQ ID NO:5. In some other embodiments, the antibodyis an IgG comprising a light chain complementarity region (CDR) of theCDRL1 sequence in SEQ ID NO:11, a CDRL2 of the CDRL2 sequence in SEQ IDNO:11, and a CDRL3 of the CDRL3 sequence in SEQ ID NO:11, and a heavychain complementarity determining region (CDR) of the CDRH1 sequence inSEQ ID NO:7, a CDRH2 of the CDRH2 sequence in SEQ ID NO:7, and a CDRH3of the CDRH3 sequence in SEQ ID NO:7. In some embodiments, the antibodyis an IgG comprising the amino acid sequences of: SEQ ID NO:20 or SEQ IDNO:21 (21B12 CDRH1), and SEQ ID NO:22 (21B12 CDRH2), and SEQ ID NO:23(21B12 CDRH3), and SEQ ID NO:24 (21B12 CDRL1), and SEQ ID NO:25 (21B12CDRL2), and SEQ ID NO:26 (21B12 CDRL3).

In the crystals or formulations described herein, the anti-PCSK9 IgGantibody can comprise the heavy and light chain variable regions of anantibody having at least 70%, at least 80%, at least 90%, at least 95%,at least 98%, at least 99% sequence identity to antibody, 21B12. Thus,in some embodiments, the antibody is an IgG comprising a light chainvariable region comprising an amino acid sequence that is at least 70%identical to that of SEQ ID NO:9 or SEQ ID NO:11 and a heavy chainvariable region that comprises an amino acid sequence that is at least70% identical to that of SEQ ID NO:5 or SEQ ID NO:7. In someembodiments, the antibody is an IgG comprising a light chain variableregion comprising an amino acid sequence that is at least 80% identicalto that of SEQ ID NO:9 or SEQ ID NO:11 and a heavy chain variable regionthat comprises an amino acid sequence that is at least 80% identical tothat of SEQ ID NO:5 or SEQ ID NO:7. In some embodiments, the antibody isan IgG comprising a light chain variable region comprising an amino acidsequence that is at least 90% identical to that of SEQ ID NO:9 or SEQ IDNO:11 and a heavy chain variable region that comprises an amino acidsequence that is at least 90% identical to that of SEQ ID NO:5 or SEQ IDNO:7. In some embodiments, the antibody is an IgG comprising a lightchain variable region comprising an amino acid sequence that is at least95% identical to that of SEQ ID NO:9 or SEQ ID NO:11 and a heavy chainvariable region that comprises an amino acid sequence that is at least95% identical to that of SEQ ID NO:5 or SEQ ID NO:7. In someembodiments, the antibody is an IgG comprising a light chain variableregion comprising an amino acid sequence that is at least 98% identicalto that of SEQ ID NO:9 or SEQ ID NO:11 and a heavy chain variable regionthat comprises an amino acid sequence that is at least 98% identical tothat of SEQ ID NO:5 or SEQ ID NO:7. In some embodiments, the antibody isan IgG comprising a light chain variable region comprising an amino acidsequence that is at least 99% identical to that of SEQ ID NO:9 or SEQ IDNO:11 and a heavy chain variable region that comprises an amino acidsequence that is at least 99% identical to that of SEQ ID NO:5 or SEQ IDNO:7. In some embodiments, the antibody is an IgG comprising a lightchain variable region comprising the amino acid sequence of SEQ ID NO:9or SEQ ID NO:11 and a heavy chain variable region that comprises anamino acid sequence of SEQ ID NO:5 or SEQ ID NO:7. In some embodiments,the antibody comprises a light chain variable region comprising theamino acid sequence of SEQ ID NO:9 and a heavy chain variable regionthat comprises an amino acid sequence of SEQ ID NO:5. In someembodiments, the antibody comprises a light chain variable regioncomprising the amino acid sequence of SEQ ID NO:11 and a heavy chainvariable region that comprises an amino acid sequence of SEQ ID NO:7.

In the crystals or formulations described herein, the anti-PCSK9 IgGantibody can comprise the heavy and light chain variable regionsdescribed above that are each fused to a suitable constant region. Insome embodiments, the antibody comprises the mature heavy and lightchains of antibody 21B12, (SEQ ID NOS:16 or 17, 21B12 mature light chainand SEQ ID NOS:18 or 19, 21B12 mature heavy chain). In some embodiments,the antibody comprises SEQ ID NO:16 and SEQ ID NO:18. In someembodiments, the antibody comprises SEQ ID NO:17 and SEQ ID NO:19. Insome embodiments, the antibody comprises amino acid sequences obtainableby expressing in mammalian host cells the cDNA encoding the heavy and/orlight chain, or alternatively the heavy and/or light chain variableregions, each fused to a suitable constant region, of antibody, 21B12,as described herein. In some embodiments, the antibody binds to PCSK9 ofSEQ ID NO: 1 with a KD binding affinity of 10⁻⁷ or less (lower numbersmeaning higher binding affinity.

The antibody crystals described herein can be characterized, forexample, by size, shape, morphology, salt content, crystal packing, andother properties. In some embodiments, the crystal length ranges fromabout 10 μM to about 1000 μM, optionally with a morphology that is rodshaped, plate-shaped, block-shaped, UFO shaped, football shaped, leafshaped, wheat shaped, singlet shaped, feather-shaped, ellipsoidal,straw-shaped, chrysanthemum-shaped, or spherical or mixtures thereof.Optionally, the crystals are in clusters. The crystals are alsocharacterized by x-ray diffraction. For example, antibody 21B12 crystalsmay exhibit a rod shape, block shape, hexagonal shape, leaf sharp,surfboard shape or plate shape, or a mixture thereof, or other shapes.In some embodiments, antibody 21B12 crystals exhibited rod or hexagonalshapes.

In some or any embodiments, the antibody crystals described herein arecharacterized by the type of salt. Suitable salts for the production ofantibody 21B12 crystals include, but are not limited to, one or more ofthe following: sodium dihydrogen phosphate, di-potassium hydrogenphosphate, sodium chloride, ammonium sulfite, ammonium chloride,ammonium citrate dibasic, ammonium citrate tribasic, magnesium formatedehydrate, potassium sodium tartrate tetrahydrate, tacsimate, sodiumcitrate dihydrate, sodium acetate trihydrate, di-ammonium tartrate,potassium sodium tartrate, sodium malonate, acetate, calcium acetate,cacodylate, CHES, lithium sulfate, magnesium chloride, magnesiumsulfate, zinc acetate, cesium chloride, ammonium phosphate, sodiumphosphase, potassium phosphate, sodium fluoride, potassium iodide,sodium idodide, ammonium iodide, sodium thiocyanate, potassiumthiocyanate, sodium formate, potassium formate, ammonium formate, malicacid, succinic acid, ammonium nitrate, sodium nitrate, potassiumthiocyanate, ammonium acetate and tascsimate. For example, other salts(including hydrates) for the production of antibody 21B12 crystals caninclude other dihydrogen phosphate salts, hydrogen phosphate salts,phosphate salts, fluoride salts, chloride salts, sulfate salts, tartratesalts, tacsimate salts, citrate salts, acetate salts, malonate salts,cacodylate salts, and iodide salts, thiocyanate salts, formate salts;with, for example, monovalent (e.g. sodium, potassium, ammonium) ordivalent cations (e.g. including but not limited to zinc, magnesium,calcium).

In some or any embodiments, the antibody crystals are characterized bycrystallization additives, which can influence the crystal growth and/orshape. Suitable crystallization additives include, but are not limitedto, one or more of the following: precipitants such as PEG having amolecular weight of about 400 kD to about 20,000 kD, or about 1000 kD toabout 5000 kD (e.g., PEG3350) or 2-methyl-2,4-pentanediol (MPD),surfactants including but not limited to polysorbate 20, polysorbate 80,polysorbate 100, or Triton X 100, amino acids including but not limitedto arginine, cysteine, glycine, histidine, lysine, proline etc., alphaamino acids or n-acyl-alpha amino acids including but not limited ton-acetyl L-arginine (also known as N-alpha N-acetly L-arginine orN2-acetyl-L-arginine), short peptides, small organic molecules, organicsalts, nucleotides and carbohydrates. In some or any embodiments, thecrystals are also characterized by the process by which they areproduced, including remaining impurities. In some embodiments, theadditives (e.g., PEG, MPD, glycerol) are at about 0.1% to about 95% w/vor v/v, or about 0.1% to about 70%, or about 0.1% to about 50%, or about0.1% to about 10%, or about 10% to about 50%, or about 20%-50%, or atleast 10%, or at least 20%.

Another aspect of the invention provides methods of making the crystalsdescribed herein. In some embodiments, the method comprises combining asolution of antibody 21B12 with a crystallization reagent comprising anappropriate salt, including any of the previously described salts,and/or a crystallization additive, including any of the previouslydescribed additives. In any of the embodiments described herein, thesalt in the crystallization reagent is present at a concentration ofabout 0.1M to about 30M, optionally 0.1M to about 10M, or about 1M toabout 10M. In any of the embodiments described herein, the additives(e.g., PEG, MPD, glycerol) are present at a concentration of about 0.1%to about 95% w/v or v/v, or about 0.1% to about 70%, or about 0.1% toabout 50%, or about 0.1% to about 10%, or about 10% to about 50%, orabout 20%-50%, or at least 10%, or at least 20%.

Methods of making antibody crystals optionally further comprise removingat least a portion of the crystallization buffer (e.g., bycentrifugation) after the crystals are formed. In some embodiments, thecrystals are then placed into a solution comprising an organic additive(e.g., ethanol or isopropanol). In some embodiments, excipients (e.g.,sucrose, trehalose, sorbitol or proline) are added to the solution.

The methods of making the antibody crystals optionally further comprisethe step of drying the crystals that have formed (e.g., by air dryingthe crystals or exposing the crystals to a vacuum or nitrogen gas).

Exemplary methods for producing the antibody crystals described hereininclude vapor diffusion and batch crystallization, which are known inthe art.

Another aspect described herein are crystalline formulations (e.g.,powder crystalline and liquid crystalline formulations) and methods ofusing antibody crystals described herein to prepare medicaments, such ascrystalline formulations, for therapy of mammals including humans.Therapy of any of the conditions described herein is contemplated,optionally using any of the dosing and timing regimens described herein.The crystalline formulations comprise antibody crystals, e.g. antibody,21B12 having one or more of the properties described herein (e.g. size,length, shape, salt content, additive content, crystal packing or otherproperties).

The crystalline formulations are suitable for parenteral administration,e.g. are sterile, have endotoxin levels acceptable for parenteraladministration, e.g. <0.25 EU/mL or 0.008 EU/mg, and comprisepharmaceutically acceptable excipients. The crystalline formulations arealso preferably of high protein concentrations, e.g. at least 100 mg/ml,120 mg/ml 140 mg/mL, 150 mg/mL, 160 mg/mL, 170 mg/mL, 180 mg/mL, 190mg/mL, 200 mg/mL, 210 mg/mL, 220 mg/mL, 230 mg/mL, 240 mg/mL, 250 mg/mL,260 mg/mL, 270 mg/mL, 280 mg/mL, 290 mg/mL, 300 mg/mL, 310 mg/mL, 320mg/mL, 330 mg/mL, 340 mg/mL, 350 mg/mL, 360 mg/mL, 370 mg/mL, 380 mg/mL,390 mg/mL, 400 mg/mL, 410 mg/mL, 420 mg/mL, 430 mg/mL, 440 mg/mL, 450mg/mL, 460 mg/mL, 480 mg/mL, 500 mg/mL or higher.

In some or any embodiments, the crystal formulation comprises excipientsincluding, but not limited to amino acids, sucrose, trehalose andsorbitol, or other sugars or polyols.

In some or any embodiments, the crystalline formulations have a pHranging from about 2 to about 12, or about 6 to about 9, or about 6 to8.5, or about 7 to about 7.5 and an osmolality ranging from about 180 toabout 420 mOsm/kg, or about 200 to about 400 mOsm/kg, or about 250 toabout 350 mOsm/kg. While isotonic (250-350 mOsm/kg) and physiologic pH(about 7-7.5) is preferred, formulations may be prepared outside ofthese ranges as long as the crystals are formulated in physiologicalrelevant conditions.

Optionally, the crystalline formulation suitable for parenteraladministration (e.g., subcutaneous or intramuscular) is presented in acontainer, such as a single dose vial, multidose vial, syringe,pre-filled syringe or injection device. In some or any embodiments, thecontainer comprises a single dose of an anti-PCSK9 antibody (e.g., about100 mg to about 500 mg of anti-PCSK9 antibody). In one exemplaryembodiment, a container may contain about 100 mg or 110 mg or 120 mg 130mg or 140 mg or 150 mg 160 mg or 170 mg or 180 mg or 190 mg or 200 mg or210 mg or 220 mg 230 mg or 240 mg or 250 mg 260 mg or 270 mg or 280 mgor 290 mg or 300 mg of the crystalline formulation of anti-PCSK9antibody and would be suitable for administering a single dose of about2, 3, 4, 5 or 6 up to about 16 mg/kg body weight. In other embodiments,a container may contain about 150 mg, or about 160 mg, or about 170 mg,or about 180 mg, or about 190 mg, or about 200 mg, or about 210 mg orabout 220 mg or about 230 mg; or about 240 mg, or at about 250 mg; orabout 250-450 mg; or about 280 mg, or about 290 mg or about 300 mg, orabout 350 mg or about 360 mg; or about 420 mg or about 430 mg or about440 mg or about 450 mg; or about 500 mg to about 1200 mg; or about 550mg, or about 600 mg, or about 700 mg, or about 800 mg, or about 900 mg,or about 1000 mg, or about 1100 mg, or about 1200 mg of the crystallineformulation of anti-PCSK9 antibody. In any of such embodiments, thecontainer may be suitable for administering a single dose of about 2, 3,4, 5 or 6 up to about 16 mg/kg body weight. In any of these embodiments,the container may comprise the antibody at a high protein concentrationsuch as those described herein. In any of these embodiments, thecontainer may comprise a powdered formulation and be for reconstitutionin a volume of about 0.5-2 mL.

Also disclosed are methods of reconstituting any of the foregoingpowdered formulations comprising adding a sterile diluent to achieve ahigh protein constitution such as those described herein.

Also disclosed herein is a kit comprising such a container and a labelcomprising instructions to use the appropriate volume or amount of thecrystalline formulation necessary to achieve a dose of from about 100 mgto about 1200 mg of anti-PCSK9 antibody, or from about 2-16 mg/kg ofpatient body weight.

Also disclosed herein are crystalline formulations (e.g., powdercrystalline and/or liquid crystalline formulations) that are stable atroom temperature for at least 1 month, 3 months, 6 months, 7 months, 8months, 9 months, 10 months, 11 months, 1 year, 18 months, 2 years, 3years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years orlonger. In some embodiments, the crystalline formulation comprisesantibody 21B12 crystals and the formulation is stable at roomtemperature for at least 1 month, 3 months, 6 months, 7 months, 8months, 9 months, 10 months, 11 months, 1 year, 18 months, 2 years, 3years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years ormore.

Also described herein are methods of using the formulations describedherein to treat and/or prevent cholesterol related disorders. In someembodiments, a “cholesterol related disorder” (which includes “serumcholesterol related disorders”) includes any one or more of thefollowing: familial hypercholesterolemia, non-familialhypercholesterolemia, hyperlipidemia, heart disease, metabolic syndrome,diabetes, coronary heart disease, stroke, cardiovascular diseases,Alzheimer's disease and generally dyslipidemias, which can bemanifested, for example, by an elevated total serum cholesterol,elevated LDL, elevated triglycerides, elevated VLDL, and/or low HDL.Some non-limiting examples of primary and secondary dyslipidemias thatcan be treated using the formulations describe herein, either alone, orin combination with one or more other agents include the metabolicsyndrome, diabetes mellitus, familial combined hyperlipidemia, familialhypertriglyceridemia, familial hypercholesterolemias, includingheterozygous hypercholesterolemia, homozygous hypercholesterolemia,familial defective apoplipoprotein B-100; polygenichypercholesterolemia; remnant removal disease, hepatic lipasedeficiency; dyslipidemia secondary to any of the following: dietaryindiscretion, hypothyroidism, drugs including estrogen and progestintherapy, beta-blockers, and thiazide diuretics; nephrotic syndrome,chronic renal failure, Cushing's syndrome, primary biliary cirrhosis,glycogen storage diseases, hepatoma, cholestasis, acromegaly,insulinoma, isolated growth hormone deficiency, and alcohol-inducedhypertriglyceridemia. The formulations described herein can also beuseful in preventing or treating atherosclerotic diseases, such as, forexample, cardiovascular death, non-cardiovascular or all-cause death,coronary heart disease, coronary artery disease, peripheral arterialdisease, stroke (ischaemic and hemorrhagic), angina pectoris, orcerebrovascular disease and acute coronary syndrome, myocardialinfarction and unstable angina. In some embodiments, the formulationsdescribed here are useful in reducing the risk of: fatal and nonfatalheart attacks, fatal and non-fatal strokes, certain types of heartsurgery, hospitalization for heart failure, chest pain in patients withheart disease, and/or cardiovascular events because of established heartdisease such as prior heart attack, prior heart surgery, and/or chestpain with evidence of clogged arteries and/or transplant-relatedvascular disease. In some embodiments, the formulations described hereinare useful in preventing or reducing the cardiovascular risk due toelevated CRP or hsCRP. In some embodiments, the formulations describedherein can be used to reduce the risk of recurrent cardiovascularevents. Exemplary doses of anti-PCSK9 antibody to treat or preventcholesterol related disorders range from about 100 mg to about 1200 mg,or about 220 mg to about 450 mg, or about 280 mg to about 450 mg ofanti-PCSK9 antibody or 1 mg/kg to about 16 mg/kg, or about 3 mg/kg to 10mg/k, or about 5-7 mg/kg body weight of anti-PCSK9 antibody.

As will be appreciated by one of skill in the art, diseases or disordersthat are generally addressable (either treatable or preventable) throughthe use of statins can also benefit from the application of theformulations described herein. In addition, in some embodiments,disorders or disease that can benefit from the prevention of cholesterolsynthesis or increased LDLR expression can also be treated by theformulations described herein. In addition, as will be appreciated byone of skill in the art, the use of the formulations described hereincan be especially useful in the treatment of diabetes. Not only isdiabetes a risk factor for coronary heart disease, but insulin increasesthe expression of PCSK9. That is, people with diabetes have elevatedplasma lipid levels (which can be related to high PCSK9 levels) and canbenefit from lowering those levels. This is generally discussed in moredetail in Costet et al. (“Hepatic PCSK9 Expression is Regulated byNutritional Status via Insulin and Sterol Regulatory Element-bindingProtein 1C”, J. Biol. Chem., 281: 6211-6218, 2006), the entirety ofwhich is incorporated herein by reference.

In another aspect, described herein are methods of lowering the serumLDL cholesterol level in a mammalian subject comprising administering acrystalline formulation described herein to the mammalian subject in anamount effective to lower serum LDL cholesterol level, as compared to apredose serum LDL cholesterol level. In some embodiments, the serum LDLcholesterol level in the mammalian subject is reduced by at least about15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90% or more, as compared to a predose serum LDL cholesterol level.In some embodiments the serum LDL cholesterol level is reduced and thereduction is sustained for a period of at least about 7 days, 2 weeks, 3weeks, 4 weeks, 1 month, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 2 months, 3months or longer.

In another aspect, described herein are methods of lowering the PCSK9values in a mammalian subject comprising administering a crystallineformulation described herein to the mammalian subject in an amounteffective to lower PCSK9 values, as compared to a predose PCSK9 value.In some embodiments, the PCSK9 value in the mammalian subject is reducedby at least about 60%, 65%, 70%, 75%, 80%, 85%, 90% or more, as comparedto a predose PCSK9 value. In some embodiments the PCSK9 value is reducedand the reduction is sustained for a period of at least about 7 days, 2weeks, 3 weeks, 4 weeks, 1 month, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 2months, 3 months or longer.

In another aspect, described herein are methods of lowering the totalcholesterol level in a mammalian subject comprising administering acrystalline formulation described herein to the mammalian subject in anamount effective to lower total cholesterol level, as compared to apredose total cholesterol level. In some embodiments, the totalcholesterol level in the mammalian subject is reduced by at least about20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or more, as compared to apredose total cholesterol level. In some embodiments the totalcholesterol level is reduced and the reduction is sustained for a periodof at least about 7 days, 2 weeks, 3 weeks, 4 weeks, 1 month, 5 weeks, 6weeks, 7 weeks, 8 weeks, 2 months, 3 months or longer.

In another aspect, described herein are methods of lowering the non-HDLcholesterol level in a mammalian subject comprising administering acrystalline formulation described herein to the mammalian subject in anamount effective to lower non-HDL cholesterol level, as compared to apredose non-HDL cholesterol level. In some embodiments, the totalcholesterol level in the mammalian subject is reduced by at least about30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or more, ascompared to a predose non-HDL cholesterol level. In some embodiments thenon-HDL cholesterol level is reduced and the reduction is sustained fora period of at least about 7 days, 2 weeks, 3 weeks, 4 weeks, 1 month, 5weeks, 6 weeks, 7 weeks, 8 weeks, 2 months, 3 months or longer.

In another aspect, described herein are methods of lowering the ApoBlevel in a mammalian subject comprising administering a crystallineformulation described herein to the mammalian subject in an amounteffective to lower the ApoB level, as compared to a predose ApoB level.In some embodiments, the ApoB level in the mammalian subject is reducedby at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, or more, as compared to a predose ApoB level. In some embodimentsthe ApoB level is reduced and the reduction is sustained for a period ofat least about 7 days, 2 weeks, 3 weeks, 4 weeks, 1 month, 5 weeks, 6weeks, 7 weeks, 8 weeks, 2 months, 3 months or longer.

In another aspect, described herein are methods of lowering theLipoprotein A (“Lp(a)” level in a mammalian subject comprisingadministering a crystalline formulation described herein to themammalian subject in an amount effective to lower the Lp(a) level, ascompared to a predose Lp(a) level. In some embodiments, the Lp(a) levelin the mammalian subject is reduced by at least about 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, or more, as compared to a predoseLp(a) level. In some embodiments the Lp(a) level is reduced and thereduction is sustained for a period of at least about 7 days, 2 weeks, 3weeks, 4 weeks, 1 month, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 2 months, 3months or longer.

It should be understood that while various embodiments in thespecification are presented using “comprising” language, under variouscircumstances, a related embodiment may also be described using“consisting of” or “consisting essentially of” language. It is to benoted that the term “a” or “an”, refers to one or more, for example, “animmunoglobulin molecule,” is understood to represent one or moreimmunoglobulin molecules. As such, the terms “a” (or “an”), “one ormore,” and “at least one” can be used interchangeably herein.

It should also be understood that when describing a range of values, thecharacteristic being described could be an individual value found withinthe range. For example, “a pH from about pH 4 to about pH 6,” could be,but is not limited to, pH 4, 4.2, 4.6, 5.1, 5.5, etc. and any value inbetween such values. Additionally, “a pH from about pH 4 to about pH 6,”should not be construed to mean that the pH of a formulation in questionvaries 2 pH units in the range from pH 4 to pH 6 during storage, butrather a value may be picked in that range for the pH of the solution,and the pH remains buffered at about that pH. In some embodiments, whenthe term “about” is used, it means the recited number plus or minus 5%,10%, 15% or more of that recited number. The actual variation intendedis determinable from the context.

In any of the ranges described herein, the endpoints of the range areincluded in the range. However, the description also contemplates thesame ranges in which the lower and/or the higher endpoint is excluded.Additional features and variations of the invention will be apparent tothose skilled in the art from the entirety of this application,including the drawing and detailed description, and all such featuresare intended as aspects of the invention. Likewise, features of theinvention described herein can be re-combined into additionalembodiments that also are intended as aspects of the invention,irrespective of whether the combination of features is specificallymentioned above as an aspect or embodiment of the invention. Also, onlysuch limitations which are described herein as critical to the inventionshould be viewed as such; variations of the invention lackinglimitations which have not been described herein as critical areintended as aspects of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A depicts an amino acid sequence of the mature form of the PCSK9with the pro-domain underlined.

FIGS. 1B ₁-1B₄ depict amino acid and nucleic acid sequences of PCSK9with the pro-domain underlined and the signal sequence in bold.

FIGS. 2A and 2B depict the amino acid and nucleic acid sequences for thevariable domains of antibody, 21B12, CDRs are underlined and/or boxed.

FIG. 3 depicts the amino acid sequences for various constant domains.

FIGS. 4A and 4B depict the amino acid sequences for mature heavy chainsand mature light chains of antibody, 21B12.

DETAILED DESCRIPTION OF THE INVENTION

Described herein are crystals of anti-PCSK9 immunoglobulin type G (IgG)antibodies. In some embodiments, the crystals of anti-PCSK9immunoglobulin type G (IgG) antibodies are suitable for use incrystalline formulations for parenteral administration. In someembodiments, the crystals of anti-PCSK9 immunoglobulin type G (IgG)antibodies are suitable for purification and drug substance storage.Also described herein are methods of using such crystals of anti-PCSK9immunoglobulin type G (IgG) antibodies to prepare crystallineformulations for use as medicaments; formulations comprising highconcentrations of a crystalline anti-PCSK9 antibodies, methods of usingthese formulations for treatment, methods of administering theseformulations, e.g. subcutaneously or intramuscularly, and containers orkits comprising these formulations.

1. Antibodies in the Formulation

In some embodiments, the anti-PCSK9 antibody in the formulation ispresent at a concentration (a “high protein concentration”) of at leastabout 100 mg/ml, about 101 mg/ml, about 102 mg/ml, about 103 mg/ml,about 104 mg/ml, about 105 mg/ml, about 106 mg/ml, about 107 mg/ml,about 108 mg/ml, about 109 mg/ml, about 110 mg/ml, about 111 mg/ml,about 112 mg/ml, about 113 mg/ml, about 114 mg/ml, about 115 mg/ml,about 116 mg/ml, about 117 mg/ml, about 118 mg/ml, about 119 mg/ml,about 120 mg/ml, about 121 mg/ml, about 122 mg/ml, about 123 mg/ml,about 124 mg/ml, about 125 mg/ml, about 126 mg/ml, about 127 mg/ml,about 128 mg/ml, about 129 mg/ml, about 130 mg/ml, about 131 mg/ml,about 132 mg/ml, about 132 mg/ml, about 133 mg/ml, about 134 mg/ml,about 135 mg/ml, about 136 mg/ml, about 137 mg/ml, about 138 mg/ml,about 139 mg/ml, about 140 mg/ml, about 141 mg/ml, about 142 mg/ml,about 143 mg/ml, about 144 mg/ml, about 145 mg/ml, about 146 mg/ml,about 147 mg/ml, about 148 mg/ml, about 149 mg/ml, about 150 mg/ml,about 151 mg/ml, about 152 mg/ml, about 153 mg/ml, about 154 mg/ml,about 155 mg/ml, about 156 mg/ml, about 157 mg/ml, about 158 mg/ml,about 159 mg/ml, about 160 mg/ml, about 161 mg/ml, about 162 mg/ml,about 163 mg/ml, about 164 mg/ml, about 165 mg/ml, about 166 mg/ml,about 167 mg/ml, about 168 mg/ml, about 169 mg/ml, about 170 mg/ml,about 171 mg/ml, about 172 mg/ml, about 173 mg/ml, about 174 mg/ml,about 175 mg/ml, about 176 mg/ml, about 177 mg/ml, about 178 mg/ml,about 179 mg/ml, about 180 mg/ml, about 181 mg/ml, about 182 mg/ml,about 183 mg/ml, about 184 mg/ml, about 185 mg/ml, about 186 mg/ml,about 187 mg/ml, about 188 mg/ml, about 189 mg/ml, about 190 mg/ml,about 191 mg/ml, about 192 mg/ml, about 193 mg/ml, about 194 mg/ml,about 195 mg/ml, about 196 mg/ml, about 197 mg/ml, about 198 mg/ml,about 199 mg/ml, about 200 mg/ml, about 201 mg/ml, about 202 mg/ml,about 203 mg/ml, about 204 mg/ml, about 205 mg/ml, about 206 mg/ml,about 207 mg/ml, about 208 mg/ml, about 209 mg/ml, about 210 mg/ml,about 211 mg/ml, about 212 mg/ml, about 213 mg/ml, about 214 mg/ml,about 215 mg/ml, about 216 mg/ml, about 217 mg/ml, about 218 mg/ml,about 219 mg/ml, about 220 mg/ml, about 221 mg/ml, about 222 mg/ml,about 223 mg/ml, about 224 mg/ml, about 225 mg/ml, about 226 mg/ml,about 227 mg/ml, about 228 mg/ml, about 229 mg/ml, about 230 mg/ml,about 231 mg/ml, about 232 mg/ml, about 232 mg/ml, about 233 mg/ml,about 234 mg/ml, about 235 mg/ml, about 236 mg/ml, about 237 mg/ml,about 238 mg/ml, about 239 mg/ml, about 240 mg/ml, about 241 mg/ml,about 242 mg/ml, about 243 mg/ml, about 244 mg/ml, about 245 mg/ml,about 246 mg/ml, about 247 mg/ml, about 248 mg/ml, about 249 mg/ml,about 250 mg/ml, and may range up to e.g., about 450 mg/ml, about 440mg/ml, 430 mg/ml, 420 mg/ml, 410 mg/ml, 400 mg/ml, about 390 mg/ml,about 380 mg/ml, about 370 mg/ml, about 360 mg/ml, about 350 mg/ml,about 340 mg/ml, about 330 mg/ml, about 320 mg/ml, about 310 mg/ml,about 300 mg/ml, about 290 mg/ml, about 280 mg/ml, about 270 mg/ml, orabout 260 mg/ml. Any range featuring a combination of the foregoingendpoints is contemplated, including but not limited to: about 70 mg/mlto about 250 mg/ml, about 100 mg/ml to about 250 mg/ml, about 150 mg/mlto about 250 mg/ml, about 150 mg/ml to about 300 mg/ml, about 150 mg/mlto about 320 mg/ml or about 150 mg/ml to about 350 mg/ml.

In some embodiments, the anti-PCSK9 antibody is antibody 21B12. Antibody21B12 was previously described in U.S. Pat. No. 8,030,457, thedisclosure of which including sequence listing is incorporated herein byreference in its entirety.

The anti-PCSK9 antibody described herein binds to PCSK9 of SEQ ID NO: 1with a KD of 10⁻⁶ or less, or 10⁻⁷ or less, or 10⁻⁸ or less, or 10⁻⁹ orless (lower numbers meaning higher binding affinity). Affinity can bedetermined by any means known in the art, including via Biacoretechnology.

The term “21B12 antibody” as used herein refers to an IgG immunoglobulincomposed of two light chains and two heavy chains, wherein the lightchain comprises a light chain complementarity region (CDR) of the CDRL1sequence in SQ ID NO:9, a CDRL2 of the CDRL2 sequence in SQ ID NO:9, anda CDRL3 of the CDRL3 sequence in SQ ID NO:9, and the heavy chaincomprises a heavy chain complementarity determining region (CDR) of theCDRH1 sequence in SEQ ID NO:5, a CDRH2 of the CDRH2 sequence in SEQ IDNO:5, and a CDRH3 of the CDRH3 sequence in SEQ ID NO:5. In some otherembodiments, the antibody is an IgG comprising a light chaincomplementarity region (CDR) of the CDRL1 sequence in SEQ ID NO:11, aCDRL2 of the CDRL2 sequence in SEQ ID NO:11, and a CDRL3 of the CDRL3sequence in SEQ ID NO:11, and a heavy chain complementarity determiningregion (CDR) of the CDRH1 sequence in SEQ ID NO:7, a CDRH2 of the CDRH2sequence in SEQ ID NO:7, and a CDRH3 of the CDRH3 sequence in SEQ IDNO:7. In some embodiments, the 21B12 antibody comprises the amino acidsequences of: SEQ ID NO:24 (21B12 CDRL1) and SEQ ID NO:25 (21B12 CDRL2),and SEQ ID NO:26 (21B12 CDRL3) and SEQ ID NO:20 or SEQ ID NO:21 (21B12CDRH1), and SEQ ID NO:22 (21B12 CDRH2), and SEQ ID NO:23 (21B12 CDRH3).

In some embodiments, the anti-PCSK9 IgG antibody comprises the heavy andlight chain variable regions of an antibody having at least 70%, atleast 80%, at least 90%, at least 95%, at least 98%, at least 99%sequence identity to antibody, 21B12. Thus, in some embodiments, theantibody is an IgG comprising a light chain variable region comprisingan amino acid sequence that is at least 70% identical to that of SEQ IDNO:9 or SEQ ID NO:11 and a heavy chain variable region that comprises anamino acid sequence that is at least 70% identical to that of SEQ IDNO:5 or SEQ ID NO:7. In some embodiments, the antibody is an IgGcomprising a light chain variable region comprising an amino acidsequence that is at least 80% identical to that of SEQ ID NO:9 or SEQ IDNO:11 and a heavy chain variable region that comprises an amino acidsequence that is at least 80% identical to that of SEQ ID NO:5 or SEQ IDNO:7. In some embodiments, the antibody is an IgG comprising a lightchain variable region comprising an amino acid sequence that is at least90% identical to that of SEQ ID NO:9 or SEQ ID NO:11 and a heavy chainvariable region that comprises an amino acid sequence that is at least90% identical to that of SEQ ID NO:5 or SEQ ID NO:7. In someembodiments, the antibody is an IgG comprising a light chain variableregion comprising an amino acid sequence that is at least 95% identicalto that of SEQ ID NO:9 or SEQ ID NO:11 and a heavy chain variable regionthat comprises an amino acid sequence that is at least 95% identical tothat of SEQ ID NO:5 or SEQ ID NO:7. In some embodiments, the antibody isan IgG comprising a light chain variable region comprising an amino acidsequence that is at least 98% identical to that of SEQ ID NO:9 or SEQ IDNO:11 and a heavy chain variable region that comprises an amino acidsequence that is at least 98% identical to that of SEQ ID NO:5 or SEQ IDNO:7. In some embodiments, the antibody is an IgG comprising a lightchain variable region comprising an amino acid sequence that is at least99% identical to that of SEQ ID NO:9 or SEQ ID NO:11 and a heavy chainvariable region that comprises an amino acid sequence that is at least99% identical to that of SEQ ID NO:5 or SEQ ID NO:7.

In some embodiments, the light chain of the 21B12 antibody comprises theamino acid sequence of SEQ ID NO:9 or SEQ ID NO:1 (21B12 light chainvariable region) and the heavy chain of the 21B12 antibody comprises theamino acid sequence of SEQ ID NO:5 or SEQ ID NO:7 (21B12 heavy chainvariable domain). In some embodiments, the light chain of the 21B12antibody comprises the amino acid sequence of SEQ ID NO:9 (21B12 lightchain variable region) and heavy chain of the 21B12 antibody comprisesthe amino acid sequence of SEQ ID NO:5 (21B12 heavy chain variableregion). In some embodiments, the light chain of the 21B12 antibodycomprises the amino acid sequence of SEQ ID NO:11 (21B12 light chainvariable region) and heavy chain of the 21B12 antibody comprises theamino acid sequence of SEQ ID NO:7 (21B12 heavy chain variable region).In some embodiments, the light chain variable region is fused to a lightchain constant region, and the heavy chain variable region is fused toan IgG constant region. In some embodiments, the 21B12 antibodycomprises the heavy and/or light chain variable region of antibody21B12, SEQ ID NO:5 (21B12 heavy chain variable region) fused to a humanheavy chain constant region of isotype IgG1, 2, 3 or 4 (e.g., native,consensus or modified, and a number of modifications that are known notto affect binding are known in the art), and/or SEQ ID NO:9 (21B12 lightchain variable region) fused to a human light chain constant region(e.g., native, consensus or modified and a number of modifications thatare known not to affect binding are known in the art), or SEQ ID NO:7(21B12 heavy chain variable region) fused to a human heavy chainconstant region of isotype IgG1, 2, 3 or 4, and/or SEQ ID NO:11 (21B12light chain variable region) fused to a human light chain constantregion. In some embodiments, the antibody comprises the mature heavy andlight chains of antibody 21B12, (SEQ ID NO:16 or 17, 21B12 mature lightchain and SEQ ID NO:18 or 19, 21B12 mature heavy chain). In someembodiments, the antibody comprises SEQ ID NO:16 and SEQ ID NO:18. Insome embodiments, the antibody comprises SEQ ID NO:17 and SEQ ID NO:19.

In some embodiments, the antibody comprises amino acid sequencesobtainable by expressing in mammalian host cells the cDNA encoding theheavy and/or light chain, or alternatively the heavy and/or light chainvariable region, of antibody 21B12. The term “antibody” refers to anintact immunoglobulin, e.g. in the case of IgG a tetramericimmunoglobulin composed of two heavy chains and two light chains. (e.g.,chimeric, humanized, or human versions preferably having full lengthheavy and/or light chains, optionally with mutations within theframework or constant regions that retain the anti-PCSK9 bindingproperties).

An “isolated” antibody refers to an antibody, as that term is definedherein, that has been identified and separated from a component of itsnatural environment. Contaminant components of its natural environmentare materials that would interfere with diagnostic or therapeutic usesfor the antibody, and may include enzymes, hormones, and otherproteinaceous or nonproteinaceous solutes. In certain embodiments, theantibody will be purified (1) to greater than 95% by weight of antibody,and most preferably more than 99% by weight, (2) to a degree sufficientto obtain at least 15 residues of N-terminal or internal amino acidsequence, or (3) to homogeneity by SDS-PAGE under reducing ornonreducing conditions using Coomassie blue or, preferably, silverstain. Isolated naturally occurring antibody includes the antibody insitu within recombinant cells since at least one component of theantibody's natural environment will not be present. Ordinarily, however,isolated antibody will be prepared by at least one purification step.

A “monoclonal” antibody refers to an antibody obtained from a populationof substantially homogeneous antibodies, i.e., the individual antibodiescomprising the population are identical except for possible naturallyoccurring mutations that may be present in minor amounts, compared to a“polyclonal” antibody which refers to a mixed population of antibodiesof diverse sequence that bind diverse epitopes. The phrase “humanizedantibody” refers to an antibody derived from a sequence of a non-humanantibody, typically a rodent monoclonal antibody, which comprisesmodifications that render the sequence more human-like. Alternatively, ahumanized antibody may be derived from a chimeric antibody. The phrase“human” antibody refers to an antibody derived from human sequences,e.g. through screening libraries of human antibody genes through knowntechniques such as phage display, or produced using transgenic animalsthat have no endogenous immunoglobulin production and are engineered tocontain human immunoglobulin loci.

An “immunoglobulin G” or “native IgG antibody” is a tetramericglycoprotein. In a naturally-occurring immunoglobulin, each tetramer iscomposed of two identical pairs of polypeptide chains, each pair havingone “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). Theamino-terminal portion of each chain includes a “variable” (“V”) regionof about 100 to 110 or more amino acids primarily responsible forantigen recognition. The carboxy-terminal portion of each chain definesa constant region primarily responsible for effector function.Immunoglobulins can be assigned to different classes depending on theamino acid sequence of the constant domain of their heavy chains. Heavychains are classified as mu (μ), delta (Δ), gamma (γ), alpha (α), andepsilon (ε), and define the antibody's isotype as IgM, IgD, IgG, IgA,and IgE, respectively. Several of these may be further divided intosubclasses or isotypes, e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.Different isotypes have different effector functions; for example, IgG1and IgG3 isotypes have antibody-dependent cellular cytotoxicity (ADCC)activity. Human light chains are classified as kappa (κ) and lambda (λ)light chains. Within light and heavy chains, the variable and constantregions are joined by a “J” region of about 12 or more amino acids, withthe heavy chain also including a “D” region of about 10 more aminoacids. See generally, Fundamental immunology, Ch. 7 (Paul, W., ed., 2nded. Raven Press, N.Y. (1989)).

The term “hypervariable” region refers to amino acid residues from acomplementarity determining region or CDR (i.e., residues 24-34 (L1),50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35(H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain asdescribed by Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991)). “Framework” or FR residues are those variableregion residues other than the hypervariable region residues.

The term “variant” when used in connection with antibodies refers to apolypeptide sequence of an antibody that contains at least one aminoacid substitution, deletion, or insertion in the variable region or theportion equivalent to the variable region, provided that the variantretains the desired binding affinity or biological activity. Inaddition, the antibodies as described herein may have amino acidmodifications in the constant region to modify effector function of theantibody, including half-life or clearance, ADCC and/or CDC activity.Such modifications can enhance pharmacokinetics or enhance theeffectiveness of the antibody in treating cancer, for example. SeeShields et al., J. Biol. Chem., 276(9):6591-6604 (2001), incorporated byreference herein in its entirety. In the case of IgG1, modifications tothe constant region, particularly the hinge or CH2 region, may increaseor decrease effector function, including ADCC and/or CDC activity. Inother embodiments, an IgG2 constant region is modified to decreaseantibody-antigen aggregate formation. In the case of IgG4, modificationsto the constant region, particularly the hinge region, may reduce theformation of half-antibodies.

The term “modification” when used in connection with antibodies orpolypeptides described herein, includes but is not limited to, one ormore amino acid change (including substitutions, insertions ordeletions); chemical modifications that do not interfere withPCSK9-binding activity; covalent modification by conjugation totherapeutic or diagnostic agents; labeling (e.g., with radionuclides orvarious enzymes); covalent polymer attachment such as pegylation(derivatization with polyethylene glycol) and insertion or substitutionby chemical synthesis of non-natural amino acids. In some embodiments,modified polypeptides (including antibodies) of the invention willretain the binding properties of unmodified molecules of the invention.

The term “derivative” when used in connection with antibodies orpolypeptides of the invention refers to antibodies or polypeptides thatare covalently modified by conjugation to therapeutic or diagnosticagents, labeling (e.g., with radionuclides or various enzymes), covalentpolymer attachment such as pegylation (derivatization with polyethyleneglycol) and insertion or substitution by chemical synthesis ofnon-natural amino acids. In some embodiments, derivatives of theinvention will retain the binding properties of underivatized moleculesof the invention.

Proteins and non-protein agents may be conjugated to the antibodies bymethods that are known in the art. Conjugation methods include directlinkage, linkage via covalently attached linkers, and specific bindingpair members (e.g., avidin-biotin). Such methods include, for example,that described by Greenfield et al., Cancer Research 50, 6600-6607(1990) for the conjugation of doxorubicin and those described by Arnonet al., Adv. Exp. Med. Biol. 303, 79-90 (1991) and by Kiseleva et al.,Mol. Biol. (USSR) 25, 508-514 (1991) for the conjugation of platinumcompounds.

II. Production of Crystals, Crystal Formulations and Compositions

Polypeptide crystals are grown by controlled crystallization ofpolypeptides from aqueous solutions or from aqueous solutions containingorganic solvents or additives. Solution conditions that may becontrolled include, for example, the rate of evaporation of solvent,organic solvents or additives, the presence of appropriate co-solutesand buffers, pH, and temperature. A comprehensive review of the variousfactors affecting the crystallization of proteins has been published byMcPherson (1985, Methods Enzymol 114: 112-120). In addition, McPhersonand Gilliland (1988, J Crystal Growth, 90: 51-59) have compiledcomprehensive lists of polypeptides that have been crystallized, as wellas the conditions under which they were crystallized. A compendium ofcrystals and crystallization recipes, as well as a repository ofcoordinates of solved protein structures, is maintained by the ProteinData Bank at the Brookhaven National Laboratory (www.rcsb.org/pdb/;Bernstein et al., 1977, J Mol Biol 112: 535-542). It should be noted,however, that the conditions reported in most of the above-citedreferences have been optimized to yield, in most instances, a few large,diffraction quality crystals. Accordingly, it will be appreciated bythose of skill in the art that these conditions vary from protein toprotein, and do not provide a high yielding process for the large scaleproduction of crystals of any given polypeptide.

In general, crystals are produced by combining the polypeptide (i.e.,antibody) to be crystallized with an appropriate aqueous solvent oraqueous solvent containing appropriate crystallization agents, such assalts or organic solvents or additives (collectively the“crystallization reagent”). The solvent is combined with the polypeptideand may be subjected to agitation at a temperature determinedexperimentally to be appropriate for the induction of crystallizationand acceptable for the maintenance of polypeptide activity andstability. Laboratory-scale methods for crystallization include hangingdrop vapor diffusion, sitting drop vapor diffusion, microdialysis,microbatch, under oil, in gel and sandwich drop methods. The solvent canoptionally include co-crystallization additives, such as precipitants,fatty acids, reducing agents, glycerol, sulfobetaine, surfactants,polyols, divalent cations, co-factors, or chaotropes, and amino acids aswell as buffer species to control pH.

“Co-crystallization additives” include compounds that facilitatecrystallization of a polypeptide and/or compounds that stabilize theprotein and protect against denaturation. Examples of co-solutes includeammonium acetate, ammonium chloride, ammonium fluoride, ammoniumformate, ammonium nitrate, ammonium phosphate, ammonium sulfate, cadmiumchloride, cadmium sulfate, calcium acetate, calcium chloride, cesiumchloride, cobaltous chloride, CH₃(CH₂) ₁₅N(CH₃)₃ ⁺ Br.⁻ (CTAB),di-ammonium citrate, di-ammonium hydrogen phosphate, di-ammoniumphosphate, di-ammonium tartrate, di-potassium phosphate, di-sodiumphosphate, di-sodium tartrate, DL-malic acid, ferric chloride,L-proline, lithium acetate, lithium chloride, lithium nitrate, lithiumsulfate, magnesium acetate, magnesium chloride, magnesium formate,magnesium nitrate, magnesium sulfate, n-acyl-alpha amino acids includingbut not limited to n-acetyl L-arginine, nickel chloride, potassiumacetate, potassium bromide, potassium chloride, potassium citrate,potassium fluoride, potassium formate, potassium nitrate, potassiumphosphate, potassium sodium tartrate, potassium sulfate, potassiumthiocyanate, sodium acetate, sodium bromide, sodium chloride, sodiumcitrate, sodium fluoride, sodium formate, sodium malonate, sodiumnitrate, sodium phosphate, sodium sulfate, sodium thiocyanate, succinicacid, tacsimate, tri-ammonium citrate, tri-lithium citrate,trimethylamine N-oxide, tri-potassium citrate, tri-sodium citrate, zincacetate, zinc sulfate, and other compounds that function to supplyco-solutes. “Crystallization” include compounds that maintain the pH ofa solution in a desired range to facilitate crystallization of apolypeptide. Examples include ACES(N-(2-acetamido)-2-aminoethanesulfonic acid), BES(N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid), Bicine(N,N-Bis(2-hydroxyethyl)glycine), BIS-TRIS(2,2-bis-(hydroxymethyl)-2,2′,2″-nitrilotriethanol), boric acid, CAPS(3-[cyclohexylamino]-1-propanesulfonic acid), citric acid, EPPS (HEPPS,4-(2-Hydroxyethyl)piperazine-1-propanesulfonic acid), Gly-Gly(NH.sub.2CH.sub.2CONHCH.sub.2COOH, glycyl-glycine), HEPES(4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid), imidazole, MES(2-morpholinoethanesulfonic acid), MOPS(3-(N-morpholino)-propanesulfonic acid), PIPES(piperazine-1,4-bis(2-ethanesulfonic acid)), potassium chloride, sodiumacetate, sodium bicarbonate, sodium phosphate monobasic (sodiumdihydrogen phosphate), sodium phosphate dibasic,TAPS(N-[tris-(hydroxymethyl)methyl]-3-aminopropanesulfonic acid),TAPSO(N-[tris(hydroxymethyl)methyl]-3-amino-2-hydroxypropanesulfon-icacid), TES (N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid),Tricine (N-[tris(hydroxymethyl)methyl]glycine), Tris-HCl, TRIZMA(2-amino-2-(hydroxymethyl)-1,3-propanediol), and other compounds thatfunction to maintain a solution at or near a specified pH.

The selection of precipitants are one factor affecting crystallization.For example, PEG products, e.g. of molecular weight 200 to 20,000 kD,can be used. PEG3350 is a long polymer precipitant or dehydrant whichworks by volume exclusion effect. Lyotropic salts, such as ammoniumsulfate, promote precipitation processes, as do short-chain fatty acids,such as caprylic acid. Polyionic species also are useful precipitants.

Antibodies for use in formulations for subcutaneous injection, forexample, preferably are precipitated at a physiologic pH range and in acrystallization reagent that provides isotonic osmolality.

The need for additives, co-solutes, buffers, etc. and theirconcentrations are determined experimentally to facilitatecrystallization. Some examples of suitable crystallization conditionsfor a polypeptide are described in Examples 1, 2 and 3 below.

Antibody 21B12 is crystallized under a variety of conditions. Variousmorphologies of Antibody 21B12 crystals can be grown under scale-upconditions whereby the antibody in a liquid formulation is added to avolume of known crystallization reagent and stored in a sealedcontainer. Antibody 21B12 crystals can be grown under these conditionsin less than 24 hours, at room temperature and have been shown toproduce between about 30% to about 99% yield.

In an industrial-scale process, the controlled precipitation leading tocrystallization can best be carried out by the simple combination ofpolypeptide, precipitant, co-solutes and, optionally, buffers in a batchprocess. As another option, polypeptides may be crystallized by usingpolypeptide precipitates as the starting material (“seeding”). In thiscase, polypeptide precipitates are added to a crystallization solutionand incubated until crystals form. Alternative laboratorycrystallization methods, such as dialysis or vapor diffusion, can alsobe adopted. McPherson, supra and Gilliland, supra, include acomprehensive list of suitable conditions in their reviews of thecrystallization literature. Occasionally, in cases in which thecrystallized polypeptide is to be crosslinked, incompatibility betweenan intended crosslinking agent and the crystallization medium mightrequire exchanging the crystals into a more suitable solvent system.

According to some embodiments, polypeptide crystals, crystalformulations and compositions are prepared by the following process:first, the polypeptide is crystallized. Next, excipients or ingredientsas described herein are added directly to the mother liquor.Alternatively, the crystals are suspended in a solution of excipient orother formulary ingredients, after the mother liquor is removed, for aminimum of 1 hour to a maximum of 24 hours. The excipient concentrationis typically between about 0.01 to 30% w/w, which corresponds to apolypeptide crystal concentration of 99.99 to 70% w/w, respectively. Inone embodiment, the excipient concentration is between about 0.1 to 10%,which corresponds to a crystal concentration of 99.9 to 90% w/w,respectively. The mother liquor can be removed from the crystal slurryeither by filtration, buffer exchange, or by centrifugation.Subsequently, the crystals are washed with any isotonic injectablevehicle as long as the these vehicles do not dissolve the crystals,optionally with solutions of 50 to 100% of one or more organic solventsor additives such as, for example, ethanol, methanol, isopropanol orethyl acetate, or polyethelene glycol (PEG), either at room temperatureor at temperatures between −20° C. to 25° C. In addition, water can beused to wash the crystals. The crystals are the dried either by passinga stream of nitrogen, air, or inert gas over the crystals. Finally,micronizing of the crystals can be performed if necessary. The drying ofpolypeptide crystals is the removal of water, organic solvent oradditive, or liquid polymer by means including drying with N₂, air, orinert gases; vacuum oven drying; lyophilization; washing with a volatileorganic solvent or additive followed by evaporation of the solvent; orevaporation in a fume hood. Typically, drying is achieved when thecrystals become a free-flowing powder. Drying may be carried out bypassing a stream of gas over wet crystals. The gas may be selected fromthe group consisting of: nitrogen, argon, helium, carbon dioxide, air orcombinations thereof. The diameter of the particles achieved can be inthe range of 0.1 to 100 micrometers, or in the range of 0.2 to 10micrometers, or in the range of 10 to 50 micrometers, or in the range of0.5 to 2 micrometers. For formulations to be administered by inhalation,in one embodiment the particles formed from the polypeptide crystals arein the range of 0.5 to 1 micrometers.

According to some embodiments, when preparing protein crystals, proteincrystal formulations or compositions, enhancers, such as surfactants arenot added during crystallization. According to some other embodiments,when preparing protein crystals, protein crystal formulations orcompositions, enhancers, such as surfactants are added duringcrystallization. Excipients or ingredients are added to the motherliquor after crystallization, at a concentration of between about 1-10%w/w, alternatively at a concentration of between about 0.1-25% w/w,alternatively at a concentration of between about 0.1-50% w/w. Theseconcentrations correspond to crystal concentrations of 99-90% w/w,99.9-75% w/w and 99.9-50% w/w, respectively. The excipient or ingredientis incubated with the crystals in the mother liquor for about 0.1-3 hrs,alternatively the incubation is carried out for 0.1-12 hrs,alternatively the incubation is carried out for 0.1-24 hrs.

In some or any embodiments, the ingredient or excipient is dissolved ina solution other than the mother liquor, and the protein crystals areremoved from the mother liquor and suspended in the excipient oringredient solution. In some embodiments, the excipient or ingredientsolution (or resuspension vehicle) is a mixture of excipients oringredients or surfactants that is isotonic and injectable. In someembodiments, the excipient or ingredient solution (or resuspensionvehicle) is not a mixture of excipients or ingredients or surfactantsthat is isotonic and injectable. The ingredient or excipientconcentrations and the incubation times are the same as those describedabove.

Polypeptide Crystals

As used herein, “crystal” or “crystalline” refers to one form of thesolid state of matter, which is distinct from a second form—theamorphous solid state. Crystals display characteristic featuresincluding a lattice structure, characteristic shapes, and opticalproperties such as refractive index and birefringence. A crystalconsists of atoms arranged in a pattern that repeats periodically inthree dimensions (C. S. Barrett, Structure of Metals, 2nd ed.,McGraw-Hill, New York, 1952, p. 1). In contrast, amorphous material is anon-crystalline solid form of matter, sometimes referred to as anamorphous precipitate. Such precipitates have no molecular latticestructure characteristic of the crystalline solid state and do notdisplay birefringence or other spectroscopic characteristics typical ofthe crystalline forms of matter.

Polypeptide crystals are polypeptide molecules arranged in a crystallattice. Polypeptide crystals contain a pattern of specificpolypeptide-polypeptide interactions that are repeated periodically inthree dimensions. The polypeptide crystals of this invention are to bedistinguished from amorphous solid forms or precipitates ofpolypeptides, such as those obtained by lyophilizing a polypeptidesolution.

In polypeptide crystals, the polypeptide molecules form asymmetric unitswhich are arranged together to form symmetric units. The geometricstructure of the symmetric units of polypeptide crystals can be, forexample, cubic, hexagonal, monoclinic, orthorhombic, tetragonal,triclinic, or trigonal. The overall structure of the crystals in theirentirety can be, for example, in the form of bipyramids, cubes, needles,plates, prisms, rhomboids, rods, or spheres, or combinations thereof.Other observed forms include block-shaped, UFO shaped, football shaped,leaf shaped, wheat shaped, singlet shaped, feather-shaped, straw-shaped,chrysanthemum-shaped, spherical or mixtures thereof. In someembodiments, the crystals are observed in clusters. Crystals that are ofthe “cubic” structural class can more specifically have octadecahedralor dodecahedral crystal forms. The diameter of the crystals is definedas the Martin's diameter. It is measured as the length of the line,parallel to the ocular scale, that divides the randomly orientedcrystals into two equal projected areas. Crystals in forms such asneedles or rods will also have a maximal dimension that is referred toherein as the length of the crystal. The crystals are also characterizedby x-ray diffraction.

Testing Properties of Crystalline Polypeptides

After polypeptide crystals are formed, they can be subjected to variousanalyses to confirm their polypeptide content and to further examinetheir physical structure. For example, if necessary individual crystalscan be removed from the crystallization solution and washed with aqueousor organic solvents or additives, then dried (for example, by airdrying, by passing a stream of inert gas over the crystal, bylyophilization, or by vacuum). Crystals can be isolated, removed fromthe crystal growth drop, and then mounted for X-ray diffraction.

Crystals can also be characterized by a variety of means described inthe art. See, e.g., Basu et al., Expert Opin. Biol. Thera. 4, 301-317(2004), incorporated herein by reference in its entirety for itsdisclosure of protein crystal production and formulation procedures, andanalytical tools for characterizing crystals and their componentprotein. While powder X-ray diffraction is commonly used to identifycrystalline material, it requires very large and perfect proteincrystals and is not commonly applied to the protein microcrystalstypically used in crystalline formulations. Electron diffraction andsolid state nuclear magnetic resonance (ssNMR) can be applied tocharacterize crystals. Crystal size, shape and morphology (e.g. surfacemorphology) can be inspected, for example, by light microscopy,transmission electron microscopy, scanning electron microscopy, atomicforce microscopy, and/or light scattering (e.g. photon correlationspectroscopy or DLS, low angle laser light scattering or LAALS). Totalsurface area and porosity of crystals can also be characterized. Massspectrometry, micro-attenuated total reflectance Fourier transforminfrared spectroscopy (FTIR) and/or differential scanning calorimetry(DSC) can provide information about protein primary and secondarystructure.

As another example, polypeptide crystals can be removed fromcrystallization solution and washed or rinsed, or the majority ofcrystallization solution can be removed from the crystals and replacedwith a different solution. In this way, the particular salt that wasusing in the crystallization procedure can be replaced in the crystallattice with a different salt. In one embodiment of the invention,crystallized Antibody 21B12 is separated from the crystallization bufferand placed in a solution containing a salt of sodium, potassium, ormagnesium (for example, sodium acetate, sodium chloride, sodium citrate,sodium phosphate, sodium sulfate, potassium chloride, potassium citrate,or magnesium sulfate). For X-ray diffraction, the replacement solutioncan contain heavy atoms useful in determining the atomic coordinates ofthe crystallized polypeptide.

In a further example, polypeptide crystals can be removed fromcrystallization solution and solubilized in an appropriate buffer forfurther testing, such as an SDS-containing buffer for analysis of thepolypeptide that had been crystallized by gel electrophoresis. Methodsfor analysis of proteins by gel electrophoresis are well known andinclude staining a gel with silver or Coomassie blue dye, and comparingthe electrophoretic migration of the polypeptide that had beencrystallized with the migration of polypeptide markers of knownmolecular weight. In another method, the polypeptide is visualized inthe gel by use of a labeled antibody that specifically binds to thepolypeptide. Polypeptides that have been crystallized can also besolubilized in buffers appropriate for amino acid sequencing by Edmandegradation, for mass spectrometry, for other spectrographic scattering,refraction, diffraction, or absorption studies, or for labeling of thepolypeptide by attachment of a label molecule to the polypeptide.

III. Formulations for Therapeutic Administration

As used herein, the term “composition” as used herein means a mixturecomprising at least two components. In particular, described herein arecompositions comprising a crystalline anti-PCSK9 antibody, or preparedusing a crystalline anti-PCSK9 antibody. In some embodiments, thecomposition or formulation comprising or prepared using a crystallineanti-PCSK9 antibody is prepared such that it is suitable for injectionand/or administration to a patient in need thereof. Compositions to beadministered for pharmaceutical purposes to patients are substantiallysterile and do not contain any agents that are unduly toxic orinfectious to the recipient.

In some embodiments, crystalline anti-PCSK9 antibodies, such ascrystalline antibody 21B12, are administered in the form of aphysiologically acceptable composition (also referred to herein as apharmaceutical composition or as a pharmaceutical formulation)comprising a crystalline anti-PCSK9 antibody that is formulated with oneor more of the following: physiologically acceptable carriers,excipients, or diluents. Such carriers, excipients, or diluents arenontoxic to recipients at the dosages and concentrations employed.Ordinarily, the preparation of such compositions entails combining thecrystalline anti-PCSK9 antibody with one or more of the following:buffers, antioxidants such as ascorbic acid, low molecular weightpolypeptides (such as those having fewer than 10 amino acids), proteins,amino acids such as Leucine, Proline, Alanine, Valine, Glycine, Serine,Asparagine, Glutamine, Aspartic acid, Glutamic acid, Methionine,Tryptophan, Phenylalanine, Isoleucine, Threonine, Cysteine, Tyrosine,Histidine, Lysine and Arginine, carbohydrates such as glucose, sucroseor dextrins, chelating agents such as EDTA, glutathione and otherstabilizers and excipients. In liquid formulations, neutral bufferedsaline or saline mixed with nonspecific serum albumin are exemplaryappropriate diluents. In accordance with appropriate industry standards,preservatives may also be added, such as benzyl alcohol. Furtherexamples of components that may be employed in pharmaceuticalformulations are presented in Remington's Pharmaceutical Sciences,16^(th) Ed., Mack Publishing Company, Easton, Pa., 1980, and in theHandbook of Pharmaceutical Excipients, published jointly by the AmericanPharmaceutical Association and the Pharmaceutical Society of GreatBritain.

In one embodiment, it is contemplated that the formulation describedherein is prepared in a bulk formulation and as such, the components ofthe pharmaceutical composition are adjusted so that they are higher thanwould be required for administration, and are diluted appropriatelyprior to administration.

The antibody crystals described herein can be formulated as a solidcrystalline or powder formulation in forms suitable for storage andhandling, and in forms suitable for inhalation or pulmonaryadministration, for example in the form of powders for the preparationof aerosol formulations. In an further embodiment, the antibody crystalscan be formulated in a liquid solution of such crystals, or in a slurryof such crystals. In another embodiment, the antibody crystals are usedto prepare a liquid formulation, such as an aqueous formulation, fortherapeutic administration.

A. Solid Crystalline Formulations

Solid formulations of antibody crystals include crystals that have beensubstantially isolated from liquid solution or dried, and are present asfree crystals or as particles in for example powder form. In the presentcontext the expression “powder” refers to a collection of essentiallydry particles, i.e. the moisture content being below about 10% byweight, or below 6% by weight, or below 4% by weight. Polypeptidecrystals or powders can be optionally combined with carriers orsurfactants. Suitable carrier agents include 1) carbohydrates, e.g.monosaccharides such as fructose, galactose, glucose, sorbose, and thelike; 2) disaccharides, such as lactose, trehalose and the like; 3)polysaccharides, such as raffmose, maltodextrins, dextrans, and thelike; 4) alditols, such as mannitol, xylitol, and the like; 5) inorganicsalts, such as sodium chloride, and the like; and 6) organic salts, suchas sodium citrate, sodium ascorbate and the like. In certainembodiments, the carrier is selected from the group consisting oftrehalose, raffinose, mannitol, sorbitol, xylitol, inositol, sucrose,sodium chloride, and sodium citrate. Surfactants can be selected fromthe group consisting of salts of fatty acids, bile salts, phospholipidsor polysorbates. Fatty acids salts include salts of C₁₀₋₁₄ fatty acids,such as sodium caprate, sodium laurate, and sodium myristate. Bile saltsinclude salts of ursodeoxycholate, taurocholate, glycocholate, andtaurodihydrofusidate. Polysorbates include polysorbate 20 andpolysorbate 80. In one embodiment, the surfactant is a salt oftaurocholate such as sodium taurocholate. Phospholipids that can be usedas surfactants include lysophosphatidylcholine. In one embodiment, thesurfactant is polysorbate 20, and in another embodiment, the surfactantis polysorbate 80.

B. Crystals in Solution or Slurries

Also described herein is a method for rendering polypeptide crystalssuitable for storage in suspensions comprising replacing thecrystallization buffer (the mother liquor) with a non-aqueous solvent.In yet another embodiment, the crystalline slurry can be rendered solidby spinning out the first solvent and washing the remaining crystallinesolid using a second organic solvent or additive to remove water,followed by evaporation of the non-aqueous solvent. Non-aqueous slurriesof crystalline therapeutic proteins are especially useful forsubcutaneous delivery.

In one such embodiment, the polypeptide crystals described herein arecombined with liquid organic additives with the object of stabilizingthe polypeptide crystals. Such a mixture can be characterized as anaqueous-organic mixture that comprises n % organic additive, where n isbetween 1 and 99 and m % aqueous solution, where m is 100-n. Examples oforganic additives include phenolic compounds, such as m-cresol or phenolor a mixture thereof, and acetone, methyl alcohol, methyl isobutylketone, chloroform, 1-propanol, isopropanol, 2-propanol, acetonitrile,1-butanol, 2-butanol, ethyl alcohol, cyclohexane, dioxane, ethylacetate, dimethylformamide, dichloroethane, hexane, isooctane, methylenechloride, tert-butyl alcohol, toluene, carbon tetrachloride, orcombinations thereof.

C. Liquid Formulations

Another embodiment provided herein is an aqueous formulation that allowsfor stable long-term storage of a pharmaceutical composition wherein acrystalline anti-PCSK9 antibody is the active ingredient used in thepreparation of the pharmaceutical composition. This formulation isuseful, in part, because it is more convenient to use for the patient,as this formulation does not require any extra steps such asrehydrating. As used herein, a “solution” or “liquid formulation” ismeant to mean a liquid preparation that contains one or more chemicalsubstances dissolved in a suitable solvent or mixture of mutuallymiscible solvents.

Reconstitution is the dissolution of polypeptide crystals or crystalformulations or compositions in an appropriate buffer or pharmaceuticalformulation.

D. Components of Pharmaceutical Formulations

The present pharmaceutical composition is prepared by combining, inaddition to a crystalline anti-PCSK9 antibody as described above, one ormore of the following types of ingredients or excipients listed in theparagraphs below, many or all of which are available from commercialsuppliers. It will be understood by one of ordinary skill in the artthat the combining of the various components to be included in thecomposition can be done in any appropriate order, namely, the buffer canbe added first, middle or last and the tonicity modifier can also beadded first, middle or last. It is also to be understood by one ofordinary skill in the art that some of these chemicals can beincompatible in certain combinations, and accordingly, are easilysubstituted with different chemicals that have similar properties butare compatible in the relevant mixture. There is knowledge in the artregarding the suitability of various combinations of excipients andother ingredients or materials present in, for example, the containersused for storage of the pharmaceutical composition and/or the devicesused for therapeutic administration (see, for example, Akers, 2002, JPharm Sci 91: 2283-2300).

Non-limiting examples of additional agents that can be included in theformulations described herein include acidifying agents (including, butnot limited to, acetic acid, glacial acetic acid, citric acid, fumaricacid, hydrochloric acid, diluted hydrochloric acid, malic acid, nitricacid, phosphoric acid, diluted phosphoric acid, sulfuric acid, tartaricacid, and other suitable acids); active ingredients (including, but notlimited to, additional active ingredients to reduce injection sitediscomfort, and non-steroidal anti-inflammatory drugs such as, forexample, tromethamine, in an appropriate dosage); aerosol propellants(including, but not limited to, butane, dichlorodifluoromethane,dichlorotetrafluoroethane, isobutane, propane andtrichloromonofluoromethane); alcohol denaturants (including, but notlimited to, denatonium benzoate, methyl isobutyl ketone, sucroseoctacetate); alkalizing agents (including, but not limited to, strongammonia solution, ammonium carbonate, diethanolamine,diisopropanolamine, potassium hydroxide, sodium bicarbonate, sodiumborate, sodium carbonate, sodium hydroxide, trolamine); anticakingagents (including, but not limited to, calcium silicate, magnesiumsilicate, colloidal silicon dioxide and talc); antifoaming agents(including, but not limited to, dimethicone and simethicone); chelatingagents (also called sequestering agents) (including, but not limited to,edetate disodium, ethylenediaminetetraacetic acid and salts and edeticacid); coating agents (including, but not limited to, sodiumcarboxymethylcellulose, cellulose acetate, cellulose acetate phthalate,ethylcellulose, gelatin, pharmaceutical glaze, hydroxypropyl cellulose,hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate,methacrylic acid copolymer, methylcellulose, polyethylene glycol,polyvinyl acetate phthalate, shellac, sucrose, titanium dioxide,carnauba wax, microcystalline wax and zein); colors (including, but notlimited to, caramel, erythrosine (FD&C Red No. 3); FD&C Red No. 40; FD&CYellow No. 5; FD&C Yellow No. 6; FD&C Blue No. 1; red, yellow, black,blue or blends and ferric oxide); complexing agents (including, but notlimited to, ethylenediaminetetraacetic acid (EDTA) and salts thereof;edetic acid, gentisic acid ethanolmaide and oxyquinoline sulfate);desiccants (including, but not limited to. calcium chloride, calciumsulfate and silicon dioxide); filtering aids (including, but not limitedto, powdered cellulose and purified siliceous earth); flavors andperfumes (including, but not limited to, anethole, anise oil,benzaldehyde, cinnamon oil, cocoa, ethyl vanillin, menthol, methylsalicylate, monosodium glutamate, orange flower oil, orange oil,peppermint, peppermint oil, peppermint spirit, rose oil, stronger rosewater, thymol, tolu balsam tincture, vanilla, vanilla tincture andvanillin); humectants (including, but not limited to, glycerin, hexyleneglycol, propylene glycol and sorbitol); ointment bases (including, butnot limited to, lanolin, anhydrous lanolin, hydrophilic ointment, whiteointment, yellow ointment, polyethylene glycol ointment, petrolatum,hydrophilic petrolatum, white petrolatum, rose water ointment andsqualane); plasticizers (including, but not limited to, castor oil,diacetylated monoglycerides, diethyl phthalate, glycerin, mono- anddi-acetylated monoglycerides, polyethylene glycol, propylene glycol,triacetin and triethyl citrate); polymer membranes (including, but notlimited to, cellulose acetate); solvents (including, but not limited to,acetone, alcohol, diluted alcohol, amylene hydrate, benzyl benzoate,butyl alcohol, carbon tetrachloride, chloroform, corn oil, cottonseedoil, ethyl acetate, glycerin, hexylene glycol, isopropyl alcohol, methylalcohol, methylene chloride, methyl isobutyl ketone, mineral oil, peanutoil, polyethylene glycol, propylene carbonate, propylene glycol, sesameoil, water for injection, sterile water for injection, sterile water forirrigation and purified water); sorbents (including, but not limited topowdered cellulose, charcoal, purified siliceous earth; and carbondioxide sorbents: barium hydroxide lime and soda lime); stiffeningagents (including, but not limited to, hydrogenated castor oil,cetostearyl alcohol, cetyl alcohol, cetyl esters wax, hard fat,paraffin, polyethylene excipient, stearyl alcohol, emulsifying wax,white wax and yellow wax); suppository bases (including, but not limitedto, cocoa butter, hard fat and polyethylene glycol); Suspending and/orviscosity-increasing agents (including, but not limited to, acacia,agar, alginic acid, aluminum monostearate, bentonite, purifiedbentonite, magma bentonite, carbomer 934p, carboxymethylcellulosecalcium, carboxymethylcellulose sodium, carboxymethycellulose sodium 12,carrageenan, microcrystalline and carboxymethylcellulose sodiumcellulose, dextrin, gelatin, guar gum, hydroxyethyl cellulose,hydroxypropyl cellulose, hydroxypropyl methylcellulose, magnesiumaluminum silicate, methylcellulose, pectin, polyethylene oxide,polyvinyl alcohol, povidone, propylene glycol alginate, silicon dioxide,colloidal silicon dioxide, sodium alginate, tragacanth and xanthan gum);sweetening agents (including, but not limited to, aspartame, dextrates,dextrose, excipient dextrose, fructose, mannitol, saccharin, calciumsaccharin, sodium saccharin, sorbitol, solution sorbitol, sucrose,compressible sugar, confectioner's sugar and syrup); tablet binders(including, but not limited to, acacia, alginic acid, sodiumcarboxymethylcellulose, microcrystalline cellulose, dextrin,ethylcellulose, gelatin, liquid glucose, guar gum, hydroxypropylmethylcellulose, methycellulose, polyethylene oxide, povidone,pregelatinized starch and syrup); tablet and/or capsule diluents(including, but not limited to, calcium carbonate, dibasic calciumphosphate, tribasic calcium phosphate, calcium sulfate, microcrystallinecellulose, powdered cellulose, dextrates, dextrin, dextrose excipient,fructose, kaolin, lactose, mannitol, sorbitol, starch, pregelatinizedstarch, sucrose, compressible sugar and confectioner's sugar); tabletdisintegrants (including, but not limited to, alginic acid,microcrystalline cellulose, croscarmellose sodium, corspovidone,polacrilin potassium, sodium starch glycolate, starch and pregelatinizedstarch); tablet and/or capsule lubricants (including, but not limitedto, calcium stearate, glyceryl behenate, magnesium stearate, lightmineral oil, polyethylene glycol, sodium stearyl fumarate, stearic acid,purified stearic acid, talc, hydrogenated vegetable oil and zincstearate); ehicles (include, but are not limited to flavored and/orsweetened (aromatic elixir, compound benzaldehyde elixir, iso-alcoholicelixir, peppermint water, sorbitol solution, syrup, tolu balsam syrup);oleaginous (almond oil, corn oil, cottonseed oil, ethyl olcate,isopropyl myristate, isopropyl palmitate, mineral oil, light mineraloil, myristyl alcohol, octyldodecanol, olive oil, peanut oil, persicoil, seame oil, soybean oil, squalane); solid carriers such as sugarspheres; and sterile vehicles (bacteriostatic water for injection,bacteriostatic sodium chloride injection); and water-repelling agents(including, but not limited to, cyclomethicone, dimethicone andsimethicone);

Aggregation inhibitors, reduce a polypeptide's tendency to associate ininappropriate or unwanted ternary or quaternary complexes, can also beincluded in the formulations described herein. Suitable aggregationinhibitors include the amino acids L-arginine and/or, L-cysteine, whichcan act to reduce aggregation of polypeptides containing an Fc domainover long periods, e.g., two years or more. The concentration of theaggregation inhibitor in the formulation can be between about 1 mM to1M, or about 10 mM to about 200 mM, or about 10 mM to about 100 mM, orabout 15 MM to about 75 mM, or about 150 mM to about 250 mM, or about 25mM.

Antioxidants may also be included in the formulations described herein.Antioxidants contemplated for use in the preparation of the formulationsinclude amino acids such as glycine and lysine, chelating agents such asEDTA and DTPA, and free-radical scavengers such as sorbitol andmannitol. Additional antioxidants include ascorbic acid, ascorbylpalmitate, butylated hydroxyanisole, butylated hydroxytoluene,hypophosphorous acid, monothioglycerol, propyl gallate, sodiumbisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, sodiumthiosulfate, sufur dioxide, tocopherol, and tocopherols excipient. Alsocontemplated for use in inhibiting oxidation is nitrogen or carbondioxide overlay. Nitrogen or carbon dioxide overlay can be introduced tothe headspace of a vial or prefilled syringe during the filling process.

Buffering agents, which maintain the pH of the pharmaceuticalformulation in a desired range, can also be included in the formulationsdescribed herein. When the pH of the pharmaceutical composition is setat or near physiological levels, comfort of the patient uponadministration is maximized. In particular, in certain embodiments thepH of a pharmaceutical composition is within a pH range of about 4.0 to8.4, or a pH range of about 5.0 to 8.0, or a pH range of about 5.8 to7.4, or about 6.2 to 7.0. It is to be understood that the pH can beadjusted as necessary to maximize stability and solubility of thepolypeptide in a particular formulation and as such, a pH outside ofphysiological ranges, yet tolerable to the patient, is within the scopeof the invention. Various buffers suitable for use in the pharmaceuticalcomposition of the invention include histidine, alkali salts (sodium orpotassium phosphate or their hydrogen or dihydrogen salts), sodiumcitrate/citric acid, sodium acetate/acetic acid, potassium citrate,maleic acid, ammonium acetate, tris-(hydroxymethyl)-aminomethane (tris),various forms of acetate and diethanolamine, ammonium carbonate,ammonium phosphate, boric acid, lactic acid, phosphoric acid, potassiummetaphosphate, potassium phosphate monobasic, sodium lactate solution,and any other pharmaceutically acceptable pH buffering agent known inthe art. pH-adjusting agents such as hydrochloric acid, sodiumhydroxide, or a salt thereof, may also be included in order to obtainthe desired pH. One suitable buffer is sodium phosphate for maintainingpharmaceutical compositions at or near pH 6.2. In another example,acetate is a more efficient buffer at pH 5 than pH 6 so less acetate maybe used in a solution at pH 5 than at pH 6. The concentration of thebuffer in the formulation can be between about 1 mM to about 1M, orabout 10 mM to about 300 mM.

Polymeric carriers can also be included in the formulations describedherein. Polymeric carriers are polymers used for encapsulation ofpolypeptide crystals for delivery of polypeptide, including biologicaldelivery. Such polymers include biocompatible and biodegradablepolymers. The polymeric carrier may be a single polymer type or it maybe composed of a mixture of polymer types. Polymers useful as thepolymeric carrier, include for example, poly(acrylic acid),poly(cyanoacrylates), poly(amino acids), poly(anhydrides),poly(depsipeptide), poly(esters) such as poly(lactic acid) or PLA,poly(lactic-co-glycolic acid) or PLGA, poly(B-hydroxybutryate),poly(caprolactone) and poly(dioxanone); poly(ethylene glycol),poly((hydroxypropyl)methacrylamide, poly [(organo)phosphazene],poly(ortho esters), poly(vinyl alcohol), poly(vinylpyrrolidone), maleicanhydride-alkyl vinyl ether copolymers, pluronic polyols, albumin,natural and synthetic polypeptides, alginate, cellulose and cellulosederivatives, collagen, fibrin, gelatin, hyaluronic acid,oligosaccharides, glycaminoglycans, sulfated polysaccharides, or anyconventional material that will encapsulate polypeptide crystals.

Preservatives, such as antimicrobial preservatives, are alsocontemplated for use in the formulations described herein. Suitablepreservatives include, but are not limited to, benzalkonium chloride,benzalkonium chloride solution, benzelthonium chloride, benzoic acid,benzyl alcohol, butylparaben, cetylpyridinium chloride, chlorobutanol,chlorocresol, cresol, dehydroacetic acid, ethylparaben, methylparaben,methylparaben sodium, phenol, phenylethyl alcohol, phenylmercuricacetate, phenylmercuric nitrate, potassium benzoate, potassium sorbate,propylparaben, propylparaben sodium, sodium benzoate, sodiumdehydroacetate, sodium propionate, sorbic acid, thimerosal, and thymol.The amount of preservative included will be in the range of 0% to 2%(w/v) or about 1% (w/v).

Solubilizing agents and stabilizers (also referred to as emulsifyingagents, co-solutes, or co-solvents) that increase the solubility of thepolypeptide and/or stabilize the polypeptide while in solution (or indried or frozen forms) can also be added to a pharmaceuticalcomposition. Examples of solubilizing and stabilizing agents include butare not limited to sugars/polyols such as: sucrose, lactose, glycerol,xylitol, sorbitol, mannitol, maltose, inositol, trehalose, glucose;polymers such as: serum albumin (bovine serum albumin (BSA), human SA(HSA), or recombinant HA), dextran, PVA, hydroxypropyl methylcellulose(HPMC), polyethyleneimine, gelatin, polyvinylpyrrolidone (PVP),hydroxyethylcellulose (HEC); non-aqueous solvents such as: polyhydricalcohols (e.g., PEG, ethylene glycol and glycerol), dimethysulfoxide(DMSO), and dimethylformamide (DMF); amino acids such as: proline,L-methionine, L-serine, sodium glutamic acid, alanine, glycine, lysinehydrochloride, sarcosine, and gamma-aminobutyric acid; surfactants suchas: Tween-80, Tween-20, SDS, polysorbate, polyoxyethylene copolymer; andmiscellaneous stabilizing excipients such as: potassium phosphate,sodium acetate, ammonium sulfate, magnesium sulfate, sodium sulfate,trimethylamine N-oxide, betaine, metal ions (e.g., zinc, copper,calcium, manganese, and magnesium), CHAPS, monolaurate,2-O-beta-mannoglycerate; or any of the following: acacia, cholesterol,diethanolamine (adjunct), glyceryl monostearate, lanolin alcohols,lecithin, mono- and di-glycerides, monoethanolamine (adjunct), oleicacid (adjunct), oleyl alcohol (stabilizer), poloxamer, polyoxyethylene50 stearate, polyoxyl 35 caster oil, polyoxyl 40 hydrogenated castoroil, polyoxyl 10 oleyl ether, polyoxyl 20 cetostearyl ether, polyoxyl 40stearate, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate80, polysorbate 100, Triton X-100, propylene glycol diacetate, propyleneglycol monostearate, sodium lauryl sulfate, sodium stearate, sorbitanmonolaurate, sorbitan monooleate, sorbitan monopalmitate, sorbitanmonostearate, stearic acid, trolamine, emulsifying wax; wetting and/orsolubilizing agents such as benzalkonium chloride, benzethoniumchloride, cetylpyridinium chloride, docusate sodium, nonoxynol 9,nonoxynol 10, octoxynol 9, polyoxyl 50 stearate, tyloxapol; or anycombination of the above. The concentration of solubilizers/stabilizersin the formulation can be between about 0.001 to 5 weight percent, orabout 0.1 to 2 weight percent. In one embodiment, the stabilizer isselected from sorbitan mono-9-octadecenoate poly(oxy-1,2-ethanediyl)derivatives, including but not limited to, polysorbate 80 or polysorbate20. The amount of polysorbate 20 or 80 to be used in this embodiment isin the range of 0.001% to 1.0% (w/v), such as 0.005% (w/v), in singleuse or in multi-dose formulations. In another embodiment, freeL-methionine in the range of 0.05 mM to 50 mM is included in theformulation: the amount of free L-methionine is 0.05 mM to 5 mM forsingle use formulations, and 1 mM to 10 mM for multi-dose formulations.

Tonicity modifiers can also be included in the formulations describedherein. Tonicity modifiers are understood to be molecules thatcontribute to the osmolality of a solution. The osmolality of apharmaceutical composition is preferably regulated in order to maximizethe active ingredient's stability and also to minimize discomfort to thepatient upon administration. Serum is approximately 300+/−50milliosmolals per kilogram. It is generally preferred that apharmaceutical composition be isotonic with serum, i.e., having the sameor similar osmolality, which is achieved by addition of a tonicitymodifier, thus it is contemplated that the osmolality will be from about180 to about 420 milliosmolals, however, it is to be understood that theosmolality can be either higher or lower as specific conditions require.Examples of tonicity modifiers suitable for modifying osmolalityinclude, but are not limited to amino acids (e.g., arginine, cysteine,histidine and glycine), salts (e.g., sodium chloride, potassium chlorideand sodium citrate) and/or saccharides (e.g., sucrose, glucose,dextrose, glycerin, and mannitol). The concentration of the tonicitymodifier in the formulation can be between about 1 mM to 1M, or about 10mM to about 200 mM. In one embodiment, the tonicity modifier is sodiumchloride within a concentration range of 0 mM to 200 mM. In anotherembodiment, the tonicity modifier is sorbitol or trehalose and no sodiumchloride is present.

In certain embodiments, the formulation comprises a compound selectedfrom the following, or any combination thereof: salts of 1) amino acidssuch as glycine, arginine, aspartic acid, glutamic acid, lysine,asparagine, glutamine, proline; 2) carbohydrates, e.g. monosaccharidessuch as glucose, fructose, galactose, mannose, arabinose, xylose,ribose; 3) disaccharides, such as lactose, trehalose, maltose, sucrose;4) polysaccharides, such as maltodextrins, dextrans, starch, glycogen;5) alditols, such as mannitol, xylitol, lactitol, sorbitol; 6)glucuronic acid, galacturonic acid: 7) cyclodextrins, such as methylcyclodextrin, hydroxypropyl-.beta.-cyclodextrin and alike; 8) inorganicsalts, such as sodium chloride, potassium chloride, magnesium chloride,phosphates of sodium and potassium, boric acid ammonium carbonate andammonium phosphate; 9) organic salts, such as acetates, citrate,ascorbate, lactate; 10) emulsifying or solubilizing agents like acacia,diethanolamine, glyceryl monostearate, lecithin, monoethanolamine, oleicacid, oleyl alcohol, poloxamer, polysorbates, sodium lauryl sulfate,stearic acid, sorbitan monolaurate, sorbitan monostearate, and othersorbitan derivatives, polyoxyl derivatives, wax, polyoxyethylenederivatives, sorbitan derivatives; 11) viscosity increasing reagentslike, agar, alginic acid and its salts, guar gum, pectin, polyvinylalcohol, polyethylene oxide, cellulose and its derivatives propylenecarbonate, polyethylene glycol, hexylene glycol, tyloxapol; and 12)particular ingredients such as sucrose, trehalose, lactose, sorbitol,lactitol, inositol, salts of sodium and potssium such as acetate,phosphates, citrates, borate, glycine, arginine, polyethylene oxide,polyvinyl alcohol, polyethylene glycol, hexylene glycol, methoxypolyethylene glycol, gelatin, hydroxypropyl-β-cyclodextrin.

E. Sustained-Release Forms

In some embodiments, sustained-release forms (also called“controlled-release” forms) of crystalline anti-PCSK9 antibodies areused, including sustained-release forms of crystalline antibody 21B12;sustained- or controlled-release forms comprising crystalline antibody21B12, and a substance for extending the physical release or biologicalavailability of the crystalline antibody 21B12 over a desired period oftime.

Sustained-release forms suitable for use in the disclosed methodsinclude, but are not limited to, crystalline antibody 21B12 that isencapsulated in a sustained-release means such as a slowly-dissolvingbiocompatible polymer (for example, the polymeric carriers describedherein, the alginate microparticles described in U.S. Pat. No.6,036,978, or the polyethylene-vinyl acetate and poly(lactic-glucolicacid) compositions described in U.S. Pat. No. 6,083,534), admixed withsuch a polymer (including topically applied hydrogels), and or encasedin a biocompatible semi-permeable implant. Further embodiments of theinvention include additional sustained-release forms such as polymericmicroparticles, wherein a mixture of the active ingredient andsustained-release means such as polymers (for example, PLGA) aredispersed within a continuous phase, and the resulting dispersion isdirectly lyophilized to remove water and organic solvents or additivesand form said microparticles (U.S. Pat. No. 6,020,004, incorporatedherein by reference in its entirety); injectable gel compositionscomprising a biodegradable anionic polysaccharide such as an alginateester, a polypeptide, and at least one bound polyvalent metal ion (U.S.Pat. No. 6,432,449, incorporated herein by reference in its entirety);injectable biodegradable polymeric matrices having reverse thermalgelation properties and optionally pH-responsive gelation/de-gelationproperties (U.S. Pat. Nos. 6,541,033 and 6,451,346, incorporated hereinby reference in their entireties); biocompatible polyol:oil suspensions,such as those wherein the suspension comprises polyol in the range offrom about 15% to about 30% by weight (U.S. Pat. No. 6,245,740,incorporated by reference in its entirety). Such sustained release formsare suitable for continuous delivery of polypeptides throughadministration in the form of a depot, wherein the depot can be animplant, or can be in the form of injectable microspheres, nanospheres,or gels. The above listed U.S. patents (U.S. Pat. Nos. 6,036,978;6,083,534; 6,020,004; 6,432,449; 6,541,033; 6,451,346, and 6,245,740)are incorporated in their entirety by reference herein. In addition,sustained- or controlled-release forms of crystalline polypeptides ofthe invention comprise types of sustained release means such as thosedescribed in Kim, C., 2000, “Controlled Release Dosage Form Design”,Techonomic Publishing Co., Lancaster Pa., which include the following:natural polymers (gelatin, sodium alginic acid, xanthan gum, arabic gum,or chitosan), semi-synthetic polymers or cellulose derivatives(methylcellulose, ethylcellulose, hydroxyethylcellulose,hydroxypropylcellulose, hydroxyethylmethylcellulose,hydroxypropylmethylcellulose, sodium carboxymethylcellulose, celluloseacetate, cellulose acetate butyrate, cellulose acetate proprionate,cellulose acetatephthalate, or hydroxypropylmethylcellulose phthalate),and synthetic polymers (ion exchange resins (methacrylic acid,sulfonated polystyrene/divinylbenzene), polyacrylic acid (Carbopol),poly(MMA/MAA), poly(MMA/DEAMA), poly(MMA/EA), poly(vinylacetatephthalate), poly(vinyl alcohol), poly(vinyl pyrrolidone), poly(lacticacid), poly(glycolic acid) poly(lactic/glycolic acid), polyethyleneglycol, polyethylene oxide, poly(dimethyl silicone), poly(hydroxyethylmethacrylate), poly(ethylene/vinyl acetate), poly(ethylene/vinylalcohol), polybutadiene, poly(anhydride), poly(orthoester), andpoly(glutamic acid)).

Further embodiments disclosed herein include antibody 21B12 crystalsencapsulated in at least one polymeric carrier to form microspheres byvirtue of encapsulation within the matrix of the polymeric carrier topreserve their native and biologically active tertiary structure, asdescribed in U.S. Pat. No. 6,541,606, which is incorporated in itsentirety by reference herein. Antibody 21B12 crystals or formulationsthereof to be encapsulated are suspended in a polymeric carrier such asPLGA which is dissolved in an organic solvent or additive. Suchencapsulated Antibody 21B12 crystals maintain the biological activity ofantibody 21B12 for a longer period of time than antibody 21B12 insolution when stored under comparable conditions.

IV. Kits

As an additional aspect, described herein are kits which comprise one ormore formulations described herein packaged in a manner whichfacilitates their use for administration to subjects. In one embodiment,such a kit includes a formulation described herein (e.g., a compositioncomprising any of the antibodies described therein), packaged in acontainer such as a sealed bottle, vessel, single-use or multi-use vial,prefilled syringe, or prefilled injection device, optionally with alabel affixed to the container or included in the package that describesuse of the compound or composition in practicing the method. In oneaspect, the compound or composition is packaged in a unit dosage form.The kit may further include a device suitable for administering thecomposition according to a specific route of administration. Preferably,the kit contains a label that describes use of an antibody describedherein or formulation described herein.

V. Dosages

The dosage regimen involved in a method for treating a conditiondescribed herein will be determined by the attending physician,considering various factors which modify the action of drugs, e.g. theage, condition, body weight, sex and diet of the patient, the severityof any infection, time of administration and other clinical factors. Invarious aspects, the dosage is in the range of 0.1-50 mg of apreparation of antibody per kilogram of body weight (calculating themass of the protein alone, without chemical modification). In someembodiments, the dosage is about 0.5 mg/kg to 20 mg/kg, or about 0.5-10mg/kg. In some embodiments, the dosage is about 120 mg to about 1200 mg,or about 280 to about 450 mg.

In various aspects, the dosage of an anti-PCSK9 antibody, e.g., antibody21B12, can range from at least about 100 mg to at about 1400 mg; orabout 120 mg to about 1200 mg; or about 120 mg to about 1000 mg; orabout 120 mg to about 800 mg; or about 120 mg to about 700 mg; or about120 mg to about 480 mg; or about 120 mg up to about 480 mg; or about 100mg up to about 480 mg; or about 1200 mg to about 480 mg; or about 140 mgto about 480 mg; or about 145 mg to about 480 mg; or about 150 mg toabout 480 mg; or about 160 mg to about 480 mg; or about 170 mg to about480 mg; or about 180 mg to about 480 mg or about 190 mg to about 480 mgor about 200 mg to about 480 mg; or about 210 mg to about 480 mg; orabout 220 mg to about 480 mg; or about 230 mg to about 480 mg; or about240 mg to about 480 mg; or about 250 mg to about 480 mg; or about 260 mgto about 480 mg; or about 270 mg to about 480 mg; or about 280 mg toabout 480 mg; or about 290 mg to about 480 mg; or about 300 mg to about480 mg; or about 310 mg to about 480 mg; or about 320 mg to about 480mg; or about 330 mg to about 480 mg; or about 340 mg to about 480 mg; orabout 350 mg to about 480 mg; or about 360 mg to about 480 mg; or about370 mg to about 480 mg; or about 380 mg to about 480 mg; or about 390 mgto about 480 mg; or about 400 mg to about 480 mg; or about 410 mg toabout 480 mg; or about 420 mg to about 480 mg; or about 430 mg to about480 mg; or about 440 mg to about 480 mg; or about 450 mg to about 480mg; or about 460 mg to about 480 mg; or about 470 mg to about 480 mg ofan anti-PCSK9 antibody, e.g., antibody 21B12.

In certain embodiments, the frequency of dosing will take into accountthe pharmacokinetic parameters of an anti-PCSK antibody and/or anyadditional therapeutic agents in the formulation used. In certainembodiments, a clinician will administer the formulation until a dosageis reached that achieves the desired effect. In certain embodiments, theformulation can therefore be administered as a single dose, or as two,three, four or more doses (which may or may not contain the same amountof the desired molecule) over time, or as a continuous infusion via animplantation device or catheter. The formulation can also be deliveredsubcutaneously or intravenously with a standard needle and syringe. Inaddition, with respect to subcutaneous delivery, pen delivery devices,as well as autoinjector delivery devices, have applications indelivering a pharmaceutical formulation of the present invention.Further refinement of the appropriate dosage is routinely made by thoseof ordinary skill in the art and is within the ambit of tasks routinelyperformed by them. In certain embodiments, appropriate dosages can beascertained through use of appropriate dose-response data. In someembodiments, the amount and frequency of administration can take intoaccount the desired cholesterol level (serum and/or total) to beobtained and the subject's present cholesterol level, LDL level, and/orLDLR levels, all of which can be obtained by methods that are well knownto those of skill in the art.

In some embodiments, a dosage of at least about 100 mg; or up to about110 mg; or up to about 115 mg, or up to about 120 mg; or up to about 140mg; or up to about 160 mg; or up to about 200 mg; or up to about 250 mg;or up to 280 mg; or up to 300 mg; or up to 350 mg; or up to 400 mg; orup to 420 mg of an anti-PCSK9 antibody, e.g., antibody 21B12, isadministered once every other week, (or every two weeks) (Q2W), to apatient in need thereof.

In certain other embodiments, a dosage of at least about 250 mg; or upto about 280 mg; or up to about 300 mg; or up to about 350 mg; or up toabout 400 mg; or up to about 420 mg; or up to about 450 mg; or up to 480mg of a an anti-PCSK9 antibody, e.g., antibody 21B12, is administeredonce every four weeks, (or once a month), to a patient in need thereof.

The formulations are generally administered parenterally, e.g.intravenously, subcutaneously, intramuscularly, via aerosol(intrapulmonary or inhalational administration), or via depot forlong-term release. In some embodiments, the formulation is administeredintravenously by an initial bolus followed by a continuous infusion tomaintain therapeutic circulating levels of drug product. In otherembodiments, the formulation is administered as a one-time dose. Thoseof ordinary skill in the art will readily optimize effective dosages andadministration regimens as determined by good medical practice and theclinical condition of the individual patient. The frequency of dosingwill depend on the pharmacokinetic parameters of the agents and theroute of administration. The optimal pharmaceutical formulation will bedetermined by one skilled in the art depending upon the route ofadministration and desired dosage. See for example, Remington'sPharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton,Pa. 18042) pages 1435-1712, the disclosure of which is herebyincorporated by reference. Such formulations may influence the physicalstate, stability, rate of in vivo release, and rate of in vivo clearanceof the administered agents. Depending on the route of administration, asuitable dose may be calculated according to body weight, body surfacearea or organ size. Further refinement of the calculations necessary todetermine the appropriate dosage for treatment involving each of theabove mentioned formulations is routinely made by those of ordinaryskill in the art without undue experimentation, especially in light ofthe dosage information and assays disclosed herein, as well as thepharmacokinetic data observed in the human clinical trials discussedabove. Appropriate dosages may be ascertained through use of establishedassays for determining blood level dosages in conjunction withappropriate dose-response data. The final dosage regimen will bedetermined by the attending physician, considering various factors whichmodify the action of drugs, e.g. the drug's specific activity, theseverity of the damage and the responsiveness of the patient, the age,condition, body weight, sex and diet of the patient, the severity of anyinfection, time of administration and other clinical factors. As studiesare conducted, further information will emerge regarding the appropriatedosage levels and duration of treatment for various diseases andconditions.

VI. Therapeutic Uses of the Formulation

As will be appreciated by one of skill in the art, disorders that relateto, involve, or can be influenced by varied cholesterol, LDL, LDLR,PCSK9, VLDL-C, apoprotein B (“ApoB”), lipoprotein A (“Lp(a)”),triglycerides, HDL-C, non-HDL-C, and total cholesterol levels can beaddressed by the pharmaceutical formulations of the present invention.In one aspect, the anti-PCS9 antibody formulations can be used inmethods to treat and/or prevent and/or reduce the risk of disorders thatrelate to elevated serum cholesterol levels or in which elevated serumcholesterol levels are relevant. In one aspect, the anti-PCS9 antibodyformulations can be used in methods to treat and/or prevent and/orreduce the risk of disorders that relate to elevated PCSK9 values or inwhich elevated PCSK9 values are relevant. In one aspect, the anti-PCS9antibody formulations can be used in methods to treat and/or preventand/or reduce the risk of disorders that relate to elevated totalcholesterol levels or in which elevated total cholesterol levels arerelevant. In one aspect, the anti-PCS9 antibody formulations can be usedin methods to treat and/or prevent and/or reduce the risk of disordersthat relate to elevated non-HDL cholesterol levels or in which elevatednon-HDL cholesterol levels are relevant. In one aspect, the anti-PCS9antibody formulations can be used in methods to treat and/or preventand/or reduce the risk of disorders that relate to elevated ApoB levelsor in which elevated ApoB levels are relevant. In one aspect, theanti-PCS9 antibody formulations can be used in methods to treat and/orprevent and/or reduce the risk of disorders that relate to elevatedLp(a) levels or in which elevated Lp(a) levels are relevant. In oneaspect, the anti-PCS9 antibody formulations can be used in methods totreat and/or prevent and/or reduce the risk of disorders that relate toelevated triglyceride levels or in which elevated triglyceride levelsare relevant. In one aspect, the anti-PCS9 antibody formulations can beused in methods to treat and/or prevent and/or reduce the risk ofdisorders that relate to elevated VLDL-C levels or in which elevatedVLDL-C levels are relevant.

As will be appreciated by one of skill in the art, the anti-PCS9antibody formulations of the present invention can be therapeuticallyuseful in treating and/or preventing cholesterol related disorders.Exemplary, non-limiting diseases and disorders that can be treated orprevented by the administration of the pharmaceutical formulations ofthe present invention include familial hypercholesterolemia,non-familial hypercholesterolemia, hyperlipidemia, heart disease,metabolic syndrome, diabetes, coronary heart disease, stroke,cardiovascular diseases, Alzheimer's disease and generallydyslipidemias, which can be manifested, for example, by an elevatedtotal serum cholesterol, elevated LDL, elevated triglycerides, elevatedVLDL, and/or low HDL. Some non-limiting examples of primary andsecondary dyslipidemias that can be treated using the formulationsdescribed herein, either alone, or in combination with one or more otheragents include the metabolic syndrome, diabetes mellitus, familialcombined hyperlipidemia, familial hypertriglyceridemia, familialhypercholesterolemias, including heterozygous hypercholesterolemia,homozygous hypercholesterolemia, familial defective apoplipoproteinB-100; polygenic hypercholesterolemia; remnant removal disease, hepaticlipase deficiency; dyslipidemia secondary to any of the following:dietary indiscretion, hypothyroidism, drugs including estrogen andprogestin therapy, beta-blockers, and thiazide diuretics; nephroticsyndrome, chronic renal failure, Cushing's syndrome, primary biliarycirrhosis, glycogen storage diseases, hepatoma, cholestasis, acromegaly,insulinoma, isolated growth hormone deficiency, and alcohol-inducedhypertriglyceridemia. The formulations of the present invention can alsobe useful in preventing or treating atherosclerotic diseases, such as,for example, cardiovascular death, non-cardiovascular or all-causedeath, coronary heart disease, coronary artery disease, peripheralarterial disease, stroke (ischaemic and hemorrhagic), angina pectoris,or cerebrovascular disease and acute coronary syndrome, myocardialinfarction and unstable angina. In some embodiments, the formulationsare useful in reducing the risk of: fatal and nonfatal heart attacks,fatal and non-fatal strokes, certain types of heart surgery,hospitalization for heart failure, chest pain in patients with heartdisease, and/or cardiovascular events because of established heartdisease such as prior heart attack, prior heart surgery, and/or chestpain with evidence of clogged arteries and/or transplant-relatedvascular disease. In some embodiments, the formulations are useful inpreventing or reducing the cardiovascular risk due to elevated CRP orhsCRP. In some embodiments, the formulations and methods can be used toreduce the risk of recurrent cardiovascular events.

As will be appreciated by one of skill in the art, diseases or disordersthat are generally addressable (either treatable or preventable) throughthe use of statins can also benefit from the application of formulationsof this invention. In addition, as will be appreciated by one of skillin the art, the use of formulations of this invention can be especiallyuseful in the treatment of diabetes.

In some embodiments, the formulations of the present invention areadministered to those who have diabetes mellitus, abdominal aorticaneurysm, atherosclerosis and/or peripheral vascular disease in order todecrease their serum cholesterol levels to a safer range. In someembodiments, the formulations of this invention are administered topatients at risk of developing any of the herein described disorders. Insome embodiments, the formulations of this invention are administered tosubjects that smoke, or used to smoke (i.e., former smokers), havehypertension or a familial history of early heart attacks.

The formulation need not cure the subject of the disorder. Theformulation may be used therapeutically to ameliorate, in whole or inpart, a cholesterol-related disorder or symptom thereof, or to protect,in whole or in part, against further progression of acholesterol-related disorder or symptom thereof. Indeed, the materialsand methods of the invention are particularly useful for lowering serumLDL cholesterol and maintaining the reduction in serum LDL cholesterolover a period of time.

One or more administrations of a formulation described herein may becarried out over a therapeutic period of, for example, about 2 weeks toabout 12 months (e.g., about 1 month, about 2 months, about 3 months,about 4 months, about 5 months, about 6 months, about 7 months, about 8months, about 9 months, about 10 months, or about 11 months). In someembodiments, a subject is administered one or more doses of theformulation to lower serum LDL cholesterol. The term “maintain reductionof serum LDL cholesterol” as used herein means the reduction of serumLDL cholesterol resulting the initial dose of the formulation does notfall more than about 1% to about 5% over the course of about 2 weeks,about 1 month, about 2 months, about 3 months, about 6 months, about 9months about 1 year, about 18 months, about 2 years, or over the courseof the patient's life).

In addition, it may be advantageous to administer multiple doses of theformulation or space out the administration of doses, depending on thetherapeutic regimen selected for a particular subject. The formulationcan be administered periodically over a time period of one year or less(e.g., 9 months or less, 6 months or less, or 3 months or less). In thisregard, the formulation can be administered to the human once everyabout 7 days, or 2 weeks, or 3 weeks, or 1 month, or 5 weeks, or 6weeks, or 7 weeks, or 2 months, or 9 weeks, or 10 weeks, or 11 weeks, or3 months, or 13 weeks, or 14 weeks, or 15 weeks, or 4 months, or 17weeks, or 18 weeks, or 19 weeks, or 5 months, or 21 weeks, or 22 weeks,or 23 weeks, or 6 months, or 12 months.

VII. Combination Therapy

Treatment of a pathology by combining two or more agents that target thesame pathogen or biochemical pathway sometimes results in greaterefficacy and diminished side effects relative to the use of thetherapeutically relevant dose of each agent alone. In some cases, theefficacy of the drug combination is additive (the efficacy of thecombination is approximately equal to the sum of the effects of eachdrug alone), but in other cases the effect can be synergistic (theefficacy of the combination is greater than the sum of the effects ofeach drug given alone). As used herein, the term “combination therapy”means the two compounds can be delivered in a simultaneous manner, e.g.concurrently, or wherein one of the compounds is administered first,followed by the second agent, e.g., sequentially. The desired result canbe either a subjective relief of one or more symptoms or an objectivelyidentifiable improvement in the recipient of the dosage.

In some embodiments, the formulation is administered prior to,concurrent with, or subsequent to, a standard of care therapeutic forthe treatment of decreased bone mineral density. As used herein, theterm “standard of care” refers to a treatment that is generally acceptedby clinicians for a certain type of patient diagnosed with a type ofillness. In some embodiments, the standard of care therapeutic is atleast one other cholesterol-lowering (serum and/or total bodycholesterol) agent. Exemplary agents include, but are not limited to,statins (atorvastatin, cerivastatin, fluvastatin, lovastatin,mevastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin),Nicotinic acid (Niacin) (NIACOR, NIASPAN (slow release niacin),SLO-NIACIN (slow release niacin), CORDAPTIVE (laropiprant)), Fibric acid(LOPID (Gemfibrozil), TRICOR (fenofibrate), Bile acid sequestrants(QUESTRAN (cholestyramine), colesevelam (WELCHOL), COLESTID(colestipol)), Cholesterol absorption inhibitors (ZETIA (ezetimibe)),Combining nicotinic acid with statin (ADVICOR (LOVASTATIN and NIASPAN),Combining a statin with an absorption inhibitor (VYTORIN (ZOCOR andZETIA) and/or lipid modifying agents. In some embodiments, theformulation is combined with PPAR gamma agonists, PPAR alpha/gammaagonists, squalene synthase inhibitors, CETP inhibitors,anti-hypertensives, anti-diabetic agents (such as sulphonyl ureas,insulin, GLP-1 analogs, DDPIV inhibitors, e.g., metaformin), ApoBmodulators, such as mipomersan, MTP inhibitors and/or arteriosclerosisobliterans treatments. In some embodiments, the formulation is combinedwith an agent that increases the level of LDLR protein in a subject,such as statins, certain cytokines like oncostatin M, estrogen, and/orcertain herbal ingredients such as berberine. In some embodiments, theformulation is combined with an agent that increases serum cholesterollevels in a subject (such as certain anti-psychotic agents, certain HIVprotease inhibitors, dietary factors such as high fructose, sucrose,cholesterol or certain fatty acids and certain nuclear receptor agonistsand antagonists for RXR, RAR, LXR, FXR). In some embodiments, theformulation is combined with an agent that increases the level of PCSK9in a subject, such as statins and/or insulin. The combination of the twocan allow for the undesirable side-effects of other agents to bemitigated by the formulation.

In some embodiments, the formulation is administered to a subject whentreatment of a standard of care therapeutic described herein iscontraindicated.

EXAMPLES Example 1—Crystallization of Antibody, 21B12

Antibody 21B12 in 10 mM sodium acetate, 9% sucrose, pH5.2 consisting oftwo mature heavy chains (SEQ ID NO:19) and two mature light chains (SEQID NO:17) recombinantly produced by DNA encoding each of these chainswas crystallized under a variety of conditions.

Crystallization of antibody 21B12 was achieved using 18 differentcrystallization screens (Hampton Research, Aliso Viejo, Calif. andRigaku Reagents, Bainbridge Island, Wash.), which employ a method forcrystallization of macromolecules known as ‘hanging drop’ vapordiffusion. A drop composed of a mixture of the protein and thecrystallization reagent (the “crystallization buffer” or the “motherliquor” or “crystal growth solution” or the “reservoir solution”) isdeposited on the underside of a sialanized coverslip, and then the dropon the coverslip is sealed with grease and placed over typically a 24well VDX tray with sealant (Hampton Research, Aliso Viejo, Calif.(HR3-170)) causing a vapor equilibrium with a liquid reservoir ofreagent. To achieve equilibrium, water vapor exchanges between the dropand a 1 ml reservoir solution in the well of the tray. As water leavesthe drop, the protein undergoes an increase in relative concentrationwhich may eventually lead to supersaturation. It is the supersaturationof protein that is required for crystallization to take place. Typicallythe drop contains a lower concentration of reagent than the reservoir,and typically, the drop contained half the concentration of reagent inthe reservoir, because equal volumes of sample and reagent were mixed toform the drop (this was the case for all crystallization conditionsexcept the LISS (Low Ionic Strength Screen) where the protein was 4 ul,buffer was 2 ul and the precipitant was 5 ul with a total drop volume of11 ul).

In these experiments, the initial protein concentration in the drop was1 mg/ml to about 140 mg/ml.

Some of the crystallization screens were set up in 24-well VDX trayswith sealant. Each position in the VDX tray contained 1 mL of reagentreservoir, with the reagent reservoir in each well differing incomposition from that in the other wells, to establish an array ofdiffering crystallization buffer conditions. 1-10 μL of protein wasadded to 1-10 μl of reservoir solution to form the drops. Trays wereincubated at ambient room temperature.

Some hanging drop vapor diffusion crystal trays were also set up usingthe Mosquito Crystal (TTP LabTech, Melbourn Science Park, Melbourn,Hertfordshire SG8 6EE, United Kingdom) using clear flat bottompolystyrene 96 well microplates (Catalog #9017—Corning Incorporated, OneRiverfront Plaza, Corning, N.Y. 14831, USA) with 100 ul as the wellsolution, 1:1 ratio of protein to well solution in the drop. The finaldrop volume was 600 nl. 300 nl of protein was placed on the center ofeach 96 well plate seal (TTP Labtech Catalog #4150-05100) (which wasplaced on the 96 well substrate/holder) followed by 300 nl of wellsolution. The 96 well substrate was inverted and sealed on top of the 96well plate and were stored at ambient room temperature.

Crystal trays were scanned everyday for a week and then once a weekusing Carl Zeiss Axiocam MRc Microscope equipped with software Axiovison4.0, www.zeiss.com. Crystal hits were recorded and characterized usingan in house crystal scoring system and morphology description (see Table1.1). Crystal hits were tested for protein crystals via the touch test,Izit crystal dye (Hampton Research HR4-710) test and by setting upcontrol trays for water and buffer for crystallization screens.

TABLE 1.1 Crystal Scoring System and Morphology Description. ScoreDescription Score Description Morphology Descrption MorphologyDescription 0 no precipitation 49 needles UFO UFO TM trail mix 1 lightprecipitation 50 plates FT footballs C clusters 2 med precipitation 51rods LV leaves T tiny 3 heavy precipitation 52 blocks WH wheat FW few 5bi-refringence precipitation S singlet ST straws 10 oil; biphasic FEfeathers CHR chrysanthemum 15 crystal balls SK skin DR dried

Crystallization Screens:

Antibody 21B12 was screened in a total of approximately 2000 conditionsusing 18 different crystallization screens which resulted in 16 crystalhits (since Wizard IV #13 and Wizard JCSG are the same conditions).Unless specifically noted otherwise below, the crystallization screenswere set up in 24-well VDX trays with sealant.

Antibody 21B12 was screened in the LISS (Low Ionic Strength Screen)(Hampton Research HR3-170). The Low Ionic Strength Screen (LISS) is athree part crystallization screen with 18 buffers in a pH range of 2-12,6 different percentages of precipitant PEG3350 and 24% PEG3350 asdehydrant especially designed for antibody and antibody fragmentsscreening. A total of 108 conditions were screened and antibody 21B12crystallized in 0.05M Citric acid pH5.5, 12% PEG3350 on day 1 at 10mg/ml. The crystal morphology resembled leaves.

Antibody 21B12 was screened in Index Screen (Hampton Research HR2-144).The Index screen combines strategies of the Grid screen, Sparse Matrixand Incomplete Factorial with classical, contemporary and newcrystallization reagent systems. Index Screen is formulated in zones:classic salts versus pH, neutralized organic acids, high salt and lowpolymer, high polymer and low salt, low ionic strength versus pH, PEGand salt versus pH, PEG and salt. A total of 96 conditions were screenedand antibody 21B12 crystallized in Index #31 (0.8M Potassium sodiumtartrate tetrahydrate, 0.1M Tris pH8.5, 0.5% w/v Polyethylene glycolmonomethyl ether 5000) on day 1 with crystal morphology of hexagonalblocks.

Antibody 21B12 was screened in Wizard I (Emerald Bio EB-W1-T), Wizard II(Emerald Bio EB-W2-T), Wizard III (Emerald Bio EB-W3-T) and Wizard IV(Emerald Bio EB-W4-T). The primary screen variables are buffers, saltscovering a broad range of crystallization space at pH4.5 to pH 10.5. Atotal of 192 conditions were screened and antibody 21B12 crystallized inWizard I #13 (1.26M (NH4)2SO4, 0.1M cacodylate pH6.5) and Wizard I #32(10% (w/v) PEG 3000, Na/K Phosphate pH6.2) on day 3 and on day 2,respectively, with crystal morphology of hexagonal blocks. Antibody21B12 crystallized in Wizard II #15 (1.26 M (NH4)2SO4, HEPES pH7.5), andWizard II #45 (1.26M (NH4)2SO4, MES pH6.0), on day 3 and day 3,respectively, with crystal morphology of hexagonal blocks. Antibody21B1312 crystallized in Wizard IV #13 (800 mM Succinic Acid pH7.0), andWizard IV #31 (20% (w/v) Polyacrylic acid 5100, 0.1M HEPES pH7.0, 20 mMMagnesium chloride) both on day 1 with crystal morphology of rods.

Antibody 21B12 was screened in Crystal Screen HT (Hampton ResearchHR2-130). The primary screen variables are salt, pH and precipitants(salts, polymers, volatile organics and non-volatile organics). A totalof 96 conditions were screened using Mosquito Crystal and antibody 21B12crystallized in Crystal screen HT #48 (0.1M Tris HCl pH 8.5, 2.0MAmmonium phosphate monobasic) on day 4 with crystal morphology ofhexagonal blocks.

Antibody 21B12 was screened in PEG/ION HT (Hampton Research HR2-139. Theprimary screen variables are PEG, ion type, ionic strength and pH. Thescreen combines high purity Polyethylene glycol 3350, 48 different highpurity salts, comprising both anions (sulfate, nitrate, tartrate,acetate, chloride, iodide, thiocyanate, formate, citrate, phosphate andfluoride) and cations (sodium, potassium, ammonium, lithium, magnesiumand calcium) in a relatively concentration of 0.2M which also works as apH screen from pH 4-9. A total of 96 conditions were screened usingMosquito crystal and antibody 21B12 crystallized Peg/Ion HT #73 (0.1MSodium acetate trihydrate pH7.0, 12% (w/v) Polyethylene glycol 3350) onday 4 with crystal morphology as rods.

Antibody 21B12 was screened in Wizard Precipitant Synergy I (Emerald BioEB-PS-B) and Wizard Precipitant Synergy II (Emerald Bio EB-PS-B). TheWizard Precipitant screen consists of selected combinations of at leasttwo different precipitating reagents, allowing the assessment ofcrystallization factors in five different dimensions: 8 different salts,4 different organic solvents, 4 different polyethylene glycols, 9different additives, pH range from 4.5-8.5. Each of the 64 baseformulations contains precipitants at high concentration, close to theirmaximum solubility, in order to reach or exceed the protein'sprecipitation point. Corresponding formulations with precipitantconcentrations at 67%, and 33%, but same buffer concentration ensuresfiner grid coverage of crystallization space and systematiccrystallization. A total of 192 conditions were screened using Mosquitocrystal and antibody 21B12 crystallized in Wizard Precipitant Synergy I#54 (0.66M Lithium sulfate, 0.66% (v/v) PEG400, 0.1M Tris HCl pH8.5) onday 0 with a crystal morphology of hexagonal blocks; Wizard PrecipitantSynergy I #2 (1.34M Ammonium sulfate, 1.34% (v/v) PEG400, 0.1M Sodiumacetate/Acetic acid pH5.5) on day 3 with crystal morphology of hexagonalblocks; and Wizard Precipitant Synergy I #42 (1.65% (v/v) PEG 400, 0.66MAmmonium citrate/citric acid pH7.5) on day 3 with a crystal morphologyof hexagonal blocks. Antibody 21B12 crystallized in Wizard PrecipitantSynergy II #83 (0.34% (w/v) PEG 4000, 0.67M Potassium phosphatedibasic/Sodium phosphate monobasic pH7.5) and Wizard Precipitant SynergyII #88 (2% (w/v) PEG 8000, 0.5M Ammonium citrate/Ammonium hydroxidepH8.5), both on day 3 with crystal morphology of rods.

Antibody 21B12 was screened in Wizard JCSG (Emerald Bio EB-JCSG-B). Theprimary screen variables are: buffers, salts, precipitants (includevolatile reagents, high molecular weight polymers and salts) and pH. Atotal of 96 conditions were screened using Mosquito crystal and antibody21B12 crystallized in Wizard JCSG #67 (800 mM Succinic acid pH7.0) andWizard JCSG #71 (1000 mM Succinic acid pH 7.0, 100 mM HEPES freeacid/Sodium hydroxide pH7.0, 1% (w/v) PEG 2000 MME) both on day 3 withcrystal morphology of hexagonal blocks. Wizard JCSG #67 is the same ascondition Wizard IV #13 with 800 mM Succinic acid at pH7.0.

Antibody 21B12 was screened in Wizard pH Buffer Screen (Emerald BioEB-PH-B) a screen designed to optimize crystal hit condition tosimultaneously explore the effects of pH and buffer composition ofcrystal growth. The Wizard pH buffer screen consists of 12 buffersystems at 8 pH points incrementally varied by 0.4 pH units. Antibody21B12 was screened in the Wizard pH buffer screen along with 12% PEG3350and 16% PEG3350. No crystal hits were observed with the 12% PEG3350. Twocrystal hits were observed on day 2 with 16% PEG3350. Antibody 21B12crystallized in Wizard pH Buffer Screen with 16% PEG3350 in 1M phosphate(di)/Sodium (mono) phosphate, 8.2 and 1M phosphate (di)/Sodium (mono)phosphate, 8.6.

Various morphologies of antibody 21B12 crystals can be grown underscale-up conditions whereby the antibody in a liquid formulation isadded to a volume of known crystallization reagent and stored in asealed container. Antibody 21B12 crystals can be grown under theseconditions in less than 24 hours.

The foregoing Example demonstrates that antibody 21B12 wascrystallizable under a variety of crystallization conditions, butcrystals did not form under every condition tested. Approximately 2000crystallization conditions were tested in a number of differentcommercially-available (i.e., Hampton Research, Rigaku Reagents) andproprietary screens.

Example 2—Antibody 21B12 Crystal Hits Optimization and Expansion

Certain conditions that proved successful in generating Antibody 21B12crystals as described in Example 1 were optimized and expanded asfollows:

Low Ionic Strength Screen #7 (0.05M Citric Acid pH 5.5, 12% PEG33501 at4 mg/ml:

Antibody, 21B12, was screened in LISS #7 (0.05M Citric acid pH 5.5, 4%PEG3350) at 72 mg/ml. Antibody, 21B12, crystals were observed at day1 atpH5.5 and day4 at pH5.0. The crystal morphology was blocks. Antibody,21B12, was screened in LISS #7 (0.05M Citric acid pH5.5, 8% PEG3350) at72 mg/ml. Antibody, 21B12, crystals were observed at day1 at pH5.5. Thecrystal morphology was blocks. Antibody, 21B12, was screened in LISS #7(0.05M Citric acid pH5.5, 12% PEG3350) at 72 mg/ml. Antibody, 21B12,crystals were observed at day4 at pH5.5. The crystal morphology wasrods. Antibody, 21B12, was screened in LISS #7 (0.05M Citric acid pH5.5, 16% PEG3350) at 72 mg/ml. No crystal hits were observed.

Antibody, 21B12, was also screened using the Citric Acid Stock Options(Hampton Research Catalog # HR2-104) pH2.2-6.5 in 0.1 pH unit incrementswhich accounted for 44 conditions. Antibody, 21B12, crystals wereobserved from pH4.0 to 5.4, pH5.6, pH5.7, pH5.9, pH6.3 and pH6.4 onday1. On Day2 crystals were observed for pH4.0 to pH6.5. At day3.Antibody, 21B12, crystals were observed at pH3.8 while on day4,Antibody, 21B12, crystals were observed at pH3.9. Overall, Antibody,21B12, in the Citric Acid Stock Options Kit crystallized from pH3.8 topH 6.5 in 0.1 pH unit increments.

Index Screen #31 (0.8 M Potassium Sodium Tartrate Tetrahydrate, 0.1MTris pH 8.5, 0.5% (w/v) PEG MME 5000):

Index Screen #3 was expanded by screening the potassium sodium tartratetetrahydrate concentration from 0.2M to 1.2M in 0.2M increments.Antibody, 21B12, crystals were observed on day1 at 0.8M potassium sodiumtartrate tetrahydrate with crystal morphology of hexagonal blocks.

Index Screen #31 was expanded by screening the PEG MME 5000concentration from 0% to 1.5% in 0.5% increments. Antibody, 21B12,crystals were observed on day1 at all PEG MME 5000 concentrations withcrystal morphology of hexagonal blocks.

Index Screen #31 was expanded using Tris stock options buffer kit from apH range of 7.0 to 9.0 in 0.1 pH unit increments. Antibody, 21B12,crystals were observed on day1 at the following pH with crystalmorphology of hexagonal blocks: pH 8.0 to pH 8.5 in 0.1 pH unitincrements.

Index Screen #31 was expanded by screening various molecular weight PEGs(550 to 10,000). Antibody, 21B12, crystals were observed on day 1 inevery PEG attempted, regardless of molecular weight with crystalmorphology of blocks.

Index Screen #31 was expanded by screening PEG5000 from 1% to 6%.Antibody, 21B12, crystals were observed at PEG5000 concentrations from1% to 5% on day 1 or day 2. Crystal morphology was blocks.

Index Screen #31 was expanded by screening Tris pH8.1 to pH 8.5 andtartrate from 0.6M, 0.65M, 0.7M, 0.75M, 0.8M and 0.9M. Antibody, 21B12,crystals were observed at pH 8.1 to 8.4 and tartrate concentrations from0.6M to 0.9M. Crystal morphology was primarily blocks; crystals grown atpH 8.1 and lower Tartrate concentrations were a mixture of rod and blockmorphologies.

Index Screen #31 was expanded by screening various proteinconcentrations: 3 mg/ml, 6 mg/ml, 9 mg/ml, 12 mg/ml, 15 mg/ml, 18 mg/ml,21 mg/ml, 24 mg/ml, 27 mg/ml, 30 mg/ml, 33 mg/ml, 36 mg/ml, 39 mg/ml, 42mg/ml, 45 mg/ml, 48 mg/ml, 51 mg/ml, 54 mg/ml, 57 mg/ml, 60 mg/ml, 63mg/ml, 66 mg/ml, 69 mg/ml and 72 mg/ml. Antibody, 21B12, crystals wereobserved as small crystals at the following concentrations: 12 mg/ml, 15mg/ml, 60 mg/ml, 63 mg/ml, 66 mg/ml, 69 mg/ml and 72 mg/ml. Antibody,21B12, crystals were observed as hexagonal blocks for the rest of theconcentrations. Ostwald ripening (dissolution of smaller crystals toform large crystals) was also observed for the following concentrations:18 mg/ml, 30 mg/ml and 33 mg/ml. Ostwald ripening is observed whensmaller crystals dissolve to form a large crystal.

Wizard I #13 (1.26M Ammonium Sulfate, 0.1M Cacodylate pH6.5):

Wizard I #13 was expanded by screening Cacodylate pH range 5.1-7.4 in0.1 pH unit increments. Antibody, 21B12, crystals were observed atpH6.7, pH6.9, pH7.0, pH 7.2-pH 7.4 on day1. Antibody, 21B12, crystalswere observed on day4 at pH5.1. At day7 Antibody, 21B12 crystals wereobserved at pH5.6 and pH 6.0 with hexagonal blocks as crystalmorphology.

Wizard I #13 was expanded by screening the salt concentration range ofAmmonium sulfate. A broad salt concentration range was screened, andAntibody, 21B12, crystals were observed at 1.25M and 1.5M final Ammoniumsulfate concentration with crystal morphology resembling leaves.

Wizard I #32 (10% (w/v) PEG3000, 0.1M Sodium/Potassium Phosphate pH6.2):

Antibody, 21B12, crystal hit in Wizard I #32 was further expanded byscreening using the Stock Option Phosphate kit (HR2-251) from pH 5.0-pH8.2 in 0.2 pH unit increments. Antibody, 21B12, crystallized at day2 atpH5.6 with crystal morphology resembling leaves. Antibody, 21B12,crystal hit in Wizard I #32 was further expanded by screening theconcentration of the precipitant PEG3000 from 5% to 30% and the saltconcentration of Na/K Phosphate from 0.05M to 0.2M at pH 6.2 which didnot yield in any crystal hits.

Wizard II #15 (1.26M Ammonium Sulfate, 0.1M HEPES DH7.5):

Wizard II #15 was expanded by screening using the Stock Option HEPES kit(HR2-102) from pH 6.8-pH 8.2 in 0.1 pH unit increments. Antibody, 21B12,crystals were observed at day1 at pH 7.5 and pH8.1. At day 2, Antibody,21B12, crystals were observed at all other pH units from 6.9 to pH 7.4,pH7.6 to pH 8.2 with crystal morphology of hexagonal blocks.

Wizard II #15 was expanded by screening the concentration of the saltammonium sulfate from 0.5M to 1.75M in 0.25M increments. Antibody,21B12, crystallized at day1 at 1.25M and 1.5M salt concentration withcrystal morphology of hexagonal blocks.

Wizard II #45 (1.26M Ammonium Sulfate, 0.1M MES pH6.0):

Wizard II #45 was expanded by screening using the Stock Option MES kit(HR2-243) from pH 5.2-pH 7.1 in 0.1 pH unit increments. Antibody, 21B12,crystals were observed at day1 at pH6.3. Antibody, 21B12, crystals wereobserved at day4 at the following pH units: pH5.3, pH5.9 to pH 6.2,pH6.4 to pH7.1. Antibody, 21B12, crystals were observed at day6 at thefollowing pH units: pH5.2 to pH5.8. Antibody, 21B12, crystals wereobserved in the HEPES stock options kit at all pH units from pH5.2 to pH7.1 in 0.1 pH unit increments with crystal morphology of hexagonalblocks.

Wizard II #45 was expanded using the Stock Open Sodium Acetate kit(HR2-233) from pH 3.6-5.6 in 0.1 pH unit increments. Antibody, 21B12,crystals were observed at day1 at pH 4.6-5.6 with crystal morphology ofhexagonal blocks. Ammonium sulfate concentrations were also expandedfrom 1.0M to 3.4M using the same pH screen with hexagonal blocksobserved after day in 1.0-1.2M ammonium sulfate.

Wizard II #45 was expanded by screening the salt ammonium sulfateconcentration range from 0.1M to 1.1M with 0.2M increments. No crystalswere observed for the salt concentration screening.

Wizard IV #13 and JCSG #67 (800 mM Succinic Acid pH7.0):

Wizard IV #13 and JCSG #67 was expanded by screening using the StockOption Succinic acid kit (HR2-249) from pH 4.3-pH 6.6 in 0.1 pH unitincrements. Antibody, 21B12, crystals were observed at day1 at pH6.3 topH 6.6. Antibody, 21B12, crystals were observed on day7 at pH6.2.Antibody, 21B12, crystals were observed in the Succinic acid stockoptions kits from pH units 6.2 to pH 6.6 in 0.1 pH unit increments withcrystal morphology of hexagonal blocks.

Wizard IV #13 was screened by exploring the Succinic acid concentrationrange from 0.1M to 1.1M with 0.2M increments. Antibody, 21B12, crystalswere observed at day3 at 0.7M Succinic acid with crystal morphology ofhexagonal blocks.

Wizard IV #31(20% (w/v) Polyacrylic Acid 5100, HEPES pH7.0, 20 mMMagnesium Chloride):

Wizard IV #31 was expanded by screening using the Stock Options kitNa-HEPES (HR2-231) from pH 6.8 to pH 8.2 in 0.1 pH unit increment.Antibody, 21B12, crystals were observed on day 10 at the following pH6.8to pH 7.4 in 0.1 pH unit increment, pH7.6 to pH8 with 0.1 pH unitincrements and at pH 8.2 with crystal morphology of hexagonal blocks.

Wizard IV #31 was expanded by screening the concentration range ofPolyacrylic acid 5100 from 5% to 30% in 5% increments and from 15% to20% in 1% increments. Antibody, 21B12, crystals were observed for 15%Polyacrylic acid 5100 on day1 and were observed at 19% and 20% on day 10with crystal morphology of hexagonal blocks.

Peglon HT #73 (0.1M Sodium Acetate Trihydrate pH7.0, 12% (w/v) PEG3350):

Peglon HT #73 was expanded by screening PEG3350 concentrations from 4%to 24% in 4% increments. Antibody, 21B12, crystals were observed on day1at 8% PEG3350. The crystal morphology was leaves.

Peglon HT #73 was expanded by screening the sodium acetate trihydrateconcentration from 0.1M to 1.35M in 0.25M increments. No crystals wereobserved.

Wizard Precipitant Synergy I #42 (1.65% (w/v PEG400, 0.66M AmmoniumCitrate/Citric Acid pH7.5):

Wizard Precipitant Synergy I #42 was expanded by screening Ammoniumcitrate/citric acid pH range (pH 4.5, 5.5, 7.5 and 8.5) as well asAmmonium citrate/citric acid concentration range from 0.1M to 1.1M in0.2M increments. No crystals were observed in either of the expansions.

Wizard Precipitant Synergy I #4 (0.66M Lithium Sulfate, 0.66% (v/v) PEG400, 0.1M Tris HCl H8.5):

Wizard Precipitant Synergy I #54 was expanded by screening Lithiumsulfate concentration from 0.1M to 1.25M in 0.15 increments. Antibody,21B12, crystals were observed at 0.55M, 0.65M and 0.7M on day1 and day3respectively with crystal morphology of hexagonal blocks.

Wizard precipitant Synergy I #54 was expanded by screening in Tris StockOptions Kit (HR2-100) from pH 7.0 to pH 9.0 in 0.1 pH increments.Antibody, 21B12, crystals were observed at day 1 at the following pHunits: pH 8.0 to 8.6 in 0.1 pH unit increments, pH 8.8 and pH8.9 withcrystal morphology of hexagonal blocks or rods. Antibody, 21B12,crystals were observed on day 6 at the following pH units: pH 7.0, 7.1,7.8 and 9.0 with crystal morphology of hexagonal blocks or rods.

Example 3—Batch Crystallization and % Yield

A. Antibody 21B12, (70 mg/ml) in 10 mM sodium acetate, 9% sucrose,pH15.2 was batch crystallized in 1.5 ml centrifuge tubes with a finalvolume of 100 ul. Antibody 21B12 consisted of two mature heavy chains(SEQ ID NO:19) and two mature light chains (SEQ ID NO:17) recombinantlyproduced by DNA encoding each of these chains. Equal amounts ofAntibody, 21B12, and crystal growth condition as described in Table 3.1was added in the 1.5 ml centrifuge tube, vortexed and stored at roomtemperature. Antibody, 21B12, batch crystallized on day 1 for thefollowing crystal hits: LISS7 (0.05M Citric acid pH 5.5, 8% PEG3350),LISS7 (0.05M citric acid p H5.5, 12% PEG3350), Wizard I #32 (20% (w/v)Polyacrylic acid 5100, HEPES pH7.0, 20 mM Magnesium chloride), PEGIon HT#73 (0.1M Sodium Acetate trihydrate pH7.0, 12% (w/v) PEG3350). Antibody,21B12, crystal morphology resembled leaves in all of the batchcrystallization experiments.

TABLE 3.1 Antibody Batch Day Day Day Day Day Day 21B12 in vol Day 0 Day1 Day 2 Day 3 4 7 8 9 10 11 LISS7 (0.05M 110 ul − − + + + + + + + +Citric acid pH 5.5, 4% PEG3350) LISS7 (0.05M 110 ulLight + + + + + + + + + Citric acid ppt pH 5.5, 8% PEG3350) LISS7 (0.05M110 ul Heavy Clear, + + + + + + + + Citric acid ppt Gel pH 5.5, 12%PEG3350) Index36 100 ul − − − − − − − − − − Wizard I 13 100 ul − − − − −− − − − − Wizard I 32 100 ul − + + + + + + + + + Wizard II 15 100 ul − −− − − − − − − − Wizard II 45 100 ul − − − − − − − − − − Antibody BatchDay Day Day Day Day 21B12 in vol Day 0 Day 1 Day 2 Day 3 6 7 8 9 10Wizard IV 13 100 ul − − − − − − − − − Wizard IV 31 100 ul − − − − − − −− − Crystal 100 ul − − − − − − − − − Screen HT #48 Pegion HT 100 ulBiphasic/ + + + + + + + + #73 Gel The symbol “−” means “clear”, and thesymbol “+” means “crystals”.

B. Antibody 21B12 (70 mg/ml) in 10 mM sodium acetate, 9% sucrose, pH5.2was batch crystallized in glass vials with mini stir bars at roomtemperature with a stirring speed of 250-450 rpm, as well as maximumspeed on manual stir plate which could translate in to 550-800 rpmspeeds, to a final volume of 1 ml (conditions as described in Table3.2). More batch crystallization hits were observed with Antibody 21B12with stirring as compared to the previous batch crystallization with nostirring. The crystal slurries generated via this technique werehomogenous and the crystal size was <50 um by visual inspection via amicroscope though settling was clearly observed overnight. Antibody21B12 consisted of two mature heavy chains (SEQ ID NO:19) and two maturelight chains (SEQ ID NO:17) recombinantly produced by DNA encoding eachof these chains.

Antibody 21B12 batch crystallized at day 1 for the following crystalhits: LISS7 (0.05M citric acid pH5.5, 12% PEG3350); Wizard I #32 (20%(w/v) Polyacrylic acid 5100 HEPES pH7.0, 20 mM Magnesium chloride);Wizard II #15 (1.26M (NH4)2So4, 0.1M HEPES pH 7.5; Wizard IV #13 andWizard JCSG #67 (800 mM Succinic acid pH7.0); Crystal Screen HT #48(0.1M Tris HCl pH8.5, 2M Ammonium phosphate monobasic); PEG Ion HT #73(0.01M Sodium acetate trihydrate pH7.0, 12% (w/v) PEG3350); WizardPrecipitant synergy I #42 (1.65% (v/v) PEG400, 0.66M Ammoniumcitrate/citric acid pH7.5); Wizard Precipitant Synergy II #83 (0.34%(w/v) PEG4000, 0.67 Potassium phosphate dibasic/sodium phosphate);Wizard Precipitant Synergy II #88 (2% (w/v) PEG8000, 0.5M Ammoniumcitrate/Ammonium hydroxide pH8.5); and PEGIon HT #73 (0.1M SodiumAcetate trihydrate pH7.0, 12% (w/v) PEG3350).

TABLE 3.2 Protein in crystalline Crystal Starting form % Yield atScreens Crystal Conditions [ ] mg/ml A280 nm mg/ml day 12 ObservationsLow Ionic #7 (0.05M Citric acid 120 4.216 115.784 96.5 CrystallineStrength pH 5.5 12% PEG3350) under Screen (4 mg/ml antibody wasmicroscope (LISS) used for this screening) Index #31 (0.8M Potassium 12058.09 61.91 51.6 Opalescent screen Na tartarate but no tetrahydrate,0.1M Tris crystals pH 8.5, 0.5% w/v PEG MME 5000) Wizard I #13 (1.26M(NH4)2SO4, 120 59.679 60.321 50.3 Opalescent 0.1M cacodylate pH 6.5) butno crystals #32 (10% (w/v) PEG3000, 120 16.361 103.639 86.4 Crystalline0.1M Na/K under Phosphate pH 6.2) microscope Wizard II #15 (1.26M(NH4)2SO4, 120 61.508 58.492 48.7 Crystalline 0.1M HEPES pH 7.5) undermicroscope #45 (1.26M (NH4)2SO4, 120 61.172 58.828 49.0 Opalescent 0.1MMES pH 6.0) but no crystals Wizard IV #13 (800 mM Succinic 120 54.63565.365 54.5 Crystalline Acid pH 7.0) under microscope #31 (20% (w/v) 120n/a n/a n/a Gelated Polyacrylic acid 5100, HEPES pH 7.0, 20 mM Magnesiumchloride) Crystal #48 (D12) 0.1M Tris HCl 120 75.526 44.474 37.1Crystalline Screen HT pH 8.5, 2.0M under Ammonium phosphate microscopemonobasic Peg/Ion #73 (0.1M Sodium 120 21.84 98.16 81.8 Crystalline HTacetate trihydrate under pH 7.0, 12% (w/v) microscope Polyethyleneglycol 3350) Wizard #54 (0.66M Lithium 120 62.721 57.279 47.7 OpalescentPrecipitant sulfate, 0.66% (v/v) but no Synergy I PEG400, 0.1M Tris HClcrystals pH 8.5) #2 (1.34M Ammonium 120 n/a n/a n/a Clear sulfate, 1.34%(v/v) PEG400, 0.1M Sodium acetate/Acetic acid pH 5.5) #42 (1.65% (v/v)PEG400, 120 52.881 67.119 55.9 Crystalline 0.66M Ammonium undercitrate/citric acid microscope pH 7.5) Wizard #83 (0.34% (w/v) PEG4000,120 58.117 61.883 51.6 Crystalline Precipitant 0.67M Potassium underSynergy II phosphate microscope dibasic/Sodium phosphate monobasic pH7.5) #88 (2% (w/v) PEG8000, 120 57.249 62.751 52.3 Crystalline 0.5MAmmonium citrate/ under Ammonium hydroxide microscope pH 8.5) Wizard #67(800 mM Succinic 120 n/a n/a n/a condition JCSG acid pH 7.0) same asWizard IV #13 #71 (1000 mM Succinic 120 n/a n/a n/a n/a acid pH 7.0, 100mM HEPES free acid/Sodium hydroxide pH 7.0, 1% (w/v) PEG2000 MME)

C. Antibody 21B12 (120 mg/ml) in 10 mM sodium acetate, 220 mM proline,0.010% polysorbate 80, pH5.0 was batch crystallized in glass vials withmini stir bars at room temperature with a stirring speed of 250-450 rpm,as well as maximum speed on manual stir plate which could translate into 550-800 rpm speeds, to a final volume of 1 ml. Antibody 21B12consisted of two mature heavy chains (SEQ ID NO:19) and two mature lightchains (SEQ ID NO:17) recombinantly produced by DNA encoding each ofthese chains.

Antibody 21B12 in 10 mM sodium acetate, 220 mM proline, 0.010%polysorbate 80, pH5.0 batch crystallized at day 1 using PEGIon HT (0.1Msodium acetate trihydrate pH7.0, 12% (w/v) polyethylene glycol 3350).

D. Antibody 21B12 (190 mg/ml) in 200 mM n-acetyl L-arginine, 10 mMglutamate, pH5.2 was batch crystallized in glass vials with mini stirbars at room temperature with a stirring speed of 250-450 rpm, as wellas maximum speed on manual stir plate which could translate in to550-800 rpm speeds, to a final volume of 1 ml. Antibody 21B12 consistedof two mature heavy chains (SEQ ID NO:19) and two mature light chains(SEQ ID NO:17) recombinantly produced by DNA encoding each of thesechains.

Antibody 21B12 batch crystallized at day 1 using modified PEGIon HT(0.05 M sodium acetate trihydrate pH7.0, 12% (w/v) polyethylene glycol3350).

Example 4—Creation of Isotonic Injectable Conditions

The ingredients for the PEGIon HT #73 conditions as described in Example3 above are injectable in humans, however, the osmolality for thiscondition is hypertonic measuring 457 mOsm/kg using Advanced Instrumentsmodel 3900 by TA instruments. Since the acceptable Osmolality range foran isotonic injectable drug is between about 250-350 mOsm/kg, conditionswere explored to identify conditions in the appropriate osmolalityrange. Antibody 21B12 was batch crystallized in glass vial with ministir bars at room temperature with a stirring speed of 250-450 rpm usingthe conditions as described in Table 4.1 to a final volume of 1 ml. Thesample volume used for measuring osmolality was 250 ul. The osmolalityrange is 250-350 mOsm/kg.

0.05M Sodium acetate trihydrate pH7.0, with 11% to 13% PEG3350 isisotonic and injectable. 0.6M and 0.07M sodium acetate trihydrate pH7.0,12% PEG3350 is also isotonic and injectable. The yield for 0.05M Sodiumacetate trihydrate pH7.0, 12% PEG3350 was 79%. Antibody 21B12 was scaledup in the condition from 1 ml to 50 mls on a bench scale at roomtemperature at a 1:1 ratio of protein:crystal growth solution.

TABLE 4.1 Crystal Conditions Osmolality 0.05M Sodium acetate 176 mOsm/kgtrihydrate, 8% PEG3350 0.05M Sodium acetate 198 mOms/kg trihydrate, 9%PEG3350 0.05M Sodium acetate 220 mOms/kg trihydrate, 10% PEG3350 0.05MSodium acetate 256 mOms/kg trihydrate, 11% PEG3350 0.05M Sodium acetate305 mOms/kg trihydrate, 12% PEG3350 0.05M Sodium acetate 321 mOms/kgtrihydrate, 13% PEG3350 0.06M Sodium acetate 317 mOsm/kg trihydrate, 12%PEG33500 0.07M Sodium acetate 325 mOsm/kg trihydrate, 12% PEG3350 0.08MSodium acetate 382 mOsm/kg trihydrate, 12% PEG3350 0.09M Sodium acetate396 mOsm/kg trihydrate, 12% PEG3350

0.05M Sodium acetate trihydrate pH7.0, with 11% to 13% PEG3350 wasisotonic and injectable, as was 0.6M and 0.07M Sodium acetate trihydratepH7.0, 12% PEG3350. The yield for 0.05M Sodium acetate trihydrate pH7.0,12% PEG3350 was 79%. Antibody 21B12 was scaled up in the condition from1 ml to 50 mls on a bench scale at room temperature at a 1:1 ratio ofprotein:crystal growth solution.

Example 5—Assaying Protein Content of Antibody, 21B12, Crystals

Salts are often present in the sample or countersolvent, and these saltsmay form crystals during crystallization attempts. One popular method ofdistinguishing the growth of salt crystals from the target crystals ofinterest is through exposure to a staining dye such as IZIT™,manufactured by Hampton Research of Laguna Niguel, Calif. The IZIT™ dyestains protein crystals blue, but does not stain salt crystals.

Numerous modifications and variations in the practice of the inventionare expected to occur to those of skill in the art upon consideration ofthe presently preferred embodiments thereof. Consequently, the onlylimitations which should be placed upon the scope of the invention arethose which appear in the appended claims.

All of the U.S. patents, U.S. patent application publications, U.S.patent applications, foreign patents, foreign patent applications andnon-patent publications referred to in this specification and/or listedin the Application Data Sheet, are incorporated herein by reference, intheir entireties.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention.

1. A crystal of an anti-PCSK9 IgG antibody comprising a light chaincomplementarity region (CDR) of the CDRL1 sequence in SEQ ID NO:9, aCDRL2 of the CDRL2 sequence in SEQ ID NO:9, and a CDRL3 of the CDRL3sequence in SEQ ID NO:9, and a heavy chain complementarity determiningregion (CDR) of the CDRH1 sequence in SEQ ID NO:5, a CDRH2 of the CDRH2sequence in SEQ ID NO:5, and a CDRH3 of the CDRH3 sequence in SEQ IDNO:5.
 2. The crystal of claim 1, wherein the anti-PCSK9 IgG antibodycomprises a light chain variable region that is at least 90% identicalto that of SEQ ID NO:9 or SEQ ID NO:11 and a heavy chain variable regionthat is at least 90% identical to that of SEQ ID NO:5 or SEQ ID NO:7. 3.The crystal of claim 2, wherein the anti-PCSK9 IgG antibody comprises alight chain variable region having the amino acid sequence set forth inSEQ ID NO:9 or SEQ ID NO:11 and a heavy chain variable region comprisingthe amino acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:7.
 4. Thecrystal of claim 1, wherein the crystal has a length of about 100 μM toabout 500 μM.
 5. The crystal of claim 1 wherein the crystal has a shapeselected from the group consisting of hexagonal rods and blocks.
 6. Thecrystal of claim 1, wherein the crystal comprises a salt selected fromthe group consisting of sodium dihydrogen phosphate, di-potassiumhydrogen phosphate, sodium chloride, ammonium sulfate, potassium sodiumtartrate tetrahydrate, tacsimate, sodium citrate dihydrate, sodiumacetate trihydrate, di-ammonium tartrate, sodium malonate, acetate,calcium acetate, cacodylate, CHES, lithium sulfate, magnesium chloride,zinc acetate, cesium chloride, ammonium phosphate, sodium phosphate,potassium phosphate, sodium fluoride, potassium iodide, sodium idodide,ammonium iodide, sodium thiocyanate, potassium thiocyanate, sodiumformate, potassium formate and ammonium formate.
 7. A method of making acrystal of antibody 21B12 comprising combining a solution of antibody21B12 with a crystallization reagent comprising a salt selected from thegroup consisting of sodium dihydrogen phosphate, di-potassium hydrogenphosphate, sodium chloride, ammonium sulfate, potassium sodium tartratetetrahydrate, tacsimate, sodium citrate dihydrate, sodium acetatetrihydrate, di-ammonium tartrate, sodium malonate, acetate, calciumacetate, cacodylate, CHES, lithium sulfate, magnesium chloride, zinacetate, cesium chloride, ammonium phosphate, sodium phosphate,potassium phosphate, sodium fluoride, potassium iodide, sodium idodide,ammonium iodide, sodium thiocyanate, potassium thiocyanate, sodiumformate, potassium formate and ammonium formate optionally at pH ofabout 6 to about
 8. 8. The method of claim 7, wherein the concentrationof salt in the crystallization buffer is from about 0.1M to about 10M.9. The method of claim 7, wherein the crystallization buffer furthercomprises 2-methyl-2,4-pentanediol (MPD) or polyethylene glycol (PEG).10. The method of claim 9, wherein the MPD is present at a concentrationof about 0.1% to about 50%.
 11. The method of claim 9, wherein the PEGhas a molecular weight of about 400 kDa to about 20,000 kDa.
 12. Themethod of claim 11, wherein the PEG is present at a concentration of0.1% to about 50%.
 13. The method of claim 7, further comprisingremoving at least a portion of the crystallization buffer after crystalshave formed.
 14. The method of claim 13, wherein the portion ofcrystallization buffer is removed by centrifugation.
 15. The method ofclaim 13, wherein the crystals are placed in a solution containing anorganic additive.
 16. The method of claim 15, further comprising theaddition of an excipient to the solution.
 17. The method of claim 16,wherein the excipient is selected from the group consisting of sucrose,trehalose, and sorbitol.
 18. The method of claim 15, wherein the organicadditive is ethanol or isopropanol.
 19. The method of claim 7, furthercomprising drying crystals that have formed.
 20. The method of claim 19,wherein the crystals are dried by exposure to air, or by exposure to avacuum, or by exposure to nitrogen gas.
 21. A 21B120 antibody crystalproduced by the method of claim
 7. 22. A crystalline formulation ofantibody 21B12.
 23. A liquid crystalline formulation of antibody 21B12at a protein concentration of 100 mg/ml or greater.
 24. The crystallineformulation of claim 22, wherein the crystal comprises a salt selectedfrom the group consisting of sodium dihydrogen phosphate, di-potassiumhydrogen phosphate, sodium chloride, ammonium sulfate, potassium sodiumtartrate tetrahydrate, tacsimate, sodium citrate dihydrate, sodiumdi-hydrogen phosphate, sodium acetate trihydrate, di-ammonium tartrate,sodium malonate, acetate, calcium acetate, cacodylate, CHES, lithiumsulfate, magnesium chloride, zin acetate, cesium chloride, ammoniumphosphate, sodium phosphate, potassium phosphate, sodium fluoride,potassium iodide, sodium idodide, ammonium iodide, sodium thiocyanate,potassium thiocyanate, sodium formate, potassium formate and ammoniumformate.
 25. The crystalline formulation of claim 24, wherein thecrystals have a length of about 20 μm to about 1 mm and a morphologyselected from the group consisting of hexagonal rod shapes, block shapesor a mixture thereof.
 26. The crystalline formulation of claim 22comprising a crystal of any one of claims 1-6.
 27. The crystallineformulation of claim 22 comprising at least 20% PEG3350 precipitant. 28.The crystalline formulation of claim 27, wherein the osmolality of theformulation ranges from about 180 to about 420 mOsm/kg.
 29. A containercomprising at least 100 to about 450 mg or more of 21B12 crystals forreconstitution in a volume of 0.5-2 mL.
 30. A container comprising 21B12crystalline formulation at a concentration of at least 100 mg/ml. 31.The container of any one of claims 29-30, wherein the container is avial, syringe or injection device.
 32. A method of reconstituting thecrystalline formulation of claim 22, comprising contacting the powderwith about 0.5-2 mL of sterile diluent.
 33. A method of lowering serumLDL cholesterol or treating a disorder associated with increased levelsof serum LDL cholesterol in a mammalian subject comprising administeringthe crystal or crystalline formulation of any of the preceding claims inan amount effective to lower serum LDL cholesterol levels in the subjectas compared to a predose serum LDL cholesterol level.